The Human Scavenger Receptor CD36GLYCOSYLATION STATUS AND ITS ROLE IN TRAFFICKING AND FUNCTION*□S

Received for publication, December 1, 2008, and in revised form, April 14, 2009 Published, JBC Papers in Press, April 15, 2009, DOI 10.1074/jbc.M109.007849

Sarah J. Hoosdally‡, Edward J. Andress‡, Carol Wooding‡, Catherine A. Martin‡, and Kenneth J. Linton‡§1

From the ‡Medical Research Council (MRC) Clinical Sciences Centre, Imperial College, Hammersmith Hospital Campus,London W12 0NN and the §Institute of Cell and Molecular Science, Queen Mary University of London,Barts and The London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, United Kingdom

Human CD36 is a class B scavenger receptor expressed in avariety of cell types such as macrophage and adipocytes. Thisplasma membrane glycoprotein has a wide range of ligandsincluding oxidized low density lipoprotein and long chain fattyacids which involves the receptor in diseases such as atheroscle-rosis and insulin resistance. CD36 is heavily modified post-translationally byN-linked glycosylation, and 10 putative glyco-sylation sites situated in the large extracellular loop of theprotein have been identified; however, their utilization and rolein the folding and function of the protein have not been charac-terized.Usingmass spectrometry onpurified andpeptideN-gly-cosidase F-deglycosylatedCD36 and also by comparing the elec-trophoretic mobility of different glycosylation site mutants, wehave determined that 9 of the 10 sites can be modified by glyco-sylation. Flow cytometric analysis of the different glycosylationmutants expressed inmammalian cells established that glycosy-lation is necessary for trafficking to the plasmamembrane.Min-imally glycosylated mutants that supported trafficking wereidentified and indicated the importance of carboxyl-terminalsites Asn-247, Asn-321, andAsn-417. However, unlike SRBI, noindividual site was found to be essential for proper trafficking ofCD36. Surprisingly, these minimally glycosylated mutantsappear to be predominantly core-glycosylated, indicating thatmature glycosylation is not necessary for surface expression inmammalian cells. The data also show that neither the nature northe pattern of glycosylation is relevant to binding of modifiedlow density lipoprotein.

Human CD36, originally identified in platelets as glycopro-tein IV (1), is a class B scavenger receptor localized to theplasma membrane. It is not expressed ubiquitously but is pres-ent in a variety of different cells and tissue types including epi-thelial cells (2), macrophages (3), endothelial cells of themicro-vasculature (4), and smoothmuscle (5). Its function is complex,and its involvement in different disease scenarios, such as can-cer (6), atherosclerosis (3, 7, 8), malaria (9), and insulin resist-ance (10), 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 ascavenger receptor for the uptake of oxidized LDL2 (3, 11).Under certain physiological conditions, this results in the lipidloading of macrophages at the site of tissue damage in the arte-rial wall, leading to foam cell formation and plaque develop-ment, a key early stage in the pathogenesis of atherosclerosis (8,12). In fat andmuscle cells, CD36 plays an essential role in lipidhomeostasis by uptake of long chain fatty acids (13). In this caseCD36 deficiency has been linked to disorders in lipid metabo-lism, giving rise to increased incidences of insulin resistanceand cardiomyopathies (11, 14, 15).Although much is known about the function of CD36, less is

known about its structure. CD36 has no bacterial homologuesbut is a member of a protein family that also includes themam-malian proteins LIMPII (16), CLA-1 (17), SRBI (18), and theDrosophila proteins Croquemort (19) and emp (20). Thesequence of 471 amino acids has two short hydrophobic regionsat the carboxyl and amino termini separated by a large hydro-philic region (21); however, the topology of the protein isunclear with both ditopic (22) and type I (23) topological mod-els proposed. Both are consistent in predicting that the largehydrophilic region is extracellular, which is clearly supportedby epitope mapping studies (24). The protein is heavily modi-fied post-translationally. The six extracellular cysteines, whichare highly conserved within the orthologous CD36 subfamily,have been shown to be linked by disulfide bonds in bovineCd36(25), and the remaining four cysteines, two at each terminus,are palmitoylated (26), lending credence to the ditopic topolog-ical model.CD36 is also modified by N-linked glycosylation, which

accounts for the observation that the protein migrates with anapparent molecular mass of 78–94 kDa on SDS-PAGE (4, 27)despite a theoretical mass for the polypeptide of 53 kDa.N-Linked glycosylation is a common modification of extracel-lular and secreted proteins, and defects in the glycosylationpathways lead to a wide range of serious diseases known collec-tively as congenital disorders of glycosylation (28). Glycosyla-tion can be important for correct folding of proteins (29, 30)

* This work was supported by the Medical Research Council UK and also byBritish Heart Foundation Project Grant PG/03/044/15328 (to C. A. M. andK. J. L.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

1 To whom correspondence should be addressed: Queen Mary University ofLondon, Barts and The London School of Medicine and Dentistry, Instituteof Cell and Molecular Science, 4 Newark St., London E1 2AT, UK. E-mail:kenneth.linton@ic.ac.uk.

2 The abbreviations used are: LDL, low density lipoprotein; SRBI, scavengerreceptor class B, type I; OG, n-octyl-�-D-glucopyranoside; PNGase F, pep-tide N-glycosidase F; Endo H, endoglycosidase H; Sf21, Spodoptera frugi-perda 21; non-g, non-glycosylated; Ni-NTA, nickel-nitrilotriacetic acid; BSA,bovine serum albumin; FT-ICR, Fourier transform ion cyclotron resonance;MS, mass spectroscopy; FACS, fluorescence-activated cell sorter; PBS,phosphate-buffered saline; mAb, monoclonal antibody; Q-Tof, quadru-pole-time of flight.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 24, pp. 16277–16288, June 12, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

JUNE 12, 2009 • VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16277

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

either by directly inducing and/or stabilizing the tertiary fold ofthe polypeptide (31) or via an affinity for lectin chaperones suchas calnexin or calreticulin (32). Glycosylation has also beenshown to be important for the trafficking of certain glycopro-teins through affinity for lectin transport machinery (33). Theglycosylation status of bovine Cd36 has already been deter-mined with all eight putative sites shown to be glycosylated(34). Human and bovine CD36 are 83% identical (93% whensimilar residues are included) and share 7 glycosylation sites(human has 10 putative glycosylation sites). In the relatedmouse SRBI, which is 33% identical (54% similar) to humanCD36, there are 11 putativeN-linked glycosylation sites, only 3of which are shared with the human protein. Site-directedmutagenesis of each of the 11 sites independently in SRBI in anotherwise wild type protein indicates that all are glycosylated,with two (Asn-108 and Asn-173) important for either traffick-ing or folding. Mutagenesis of either of these two residuesresulted in very little cell surface expression of the protein (35);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 (massspectrometry and gel electrophoresis) to identify unequivocallywhich glycosylation sites are occupied in human CD36. Anti-body and ligand binding studieswith thesemutant proteins alsoprovided insights into the role of glycosylation and site occu-pancy in the trafficking and function of the protein.

EXPERIMENTAL PROCEDURES

Materials—The detergent n-octyl-�-D-glucopyranoside(OG) was purchased from Merck. The nickel-NTA-agarosewas purchased from Qiagen Ltd, UK, and BODIPY AcetylatedLDL FL� (BODIPY Ac-LDL) was from Invitrogen. All the pro-tease inhibitors, fatty acid-free BSA, and tunicamycinwere pur-chased from Sigma-Aldrich. Amicon Ultra 15 (molecularweight cutoff 50) centrifugal devices were from Millipore, andpeptide N-glycosidase F (PNGase F) and endoglycosidase H(Endo H) were from New England Biolabs. The primary anti-bodies, mouse mAb1258 (Chemicon International), and ratmAb1955 (R and D Systems), both recognize folded CD36 foruse in flow cytometry, but only mAb1955 recognizes the dena-tured product after SDS-PAGE and Western analysis.Generation of Expression Vectors Encoding CD36 with a

12-Histidine, Carboxyl-terminal Tag—NcoI and BstEIIrestriction sites were introduced into the 5� and 3� ends,respectively, of the CD36 cDNA (ATCC clone MGC-14530)by mutagenic polymerase chain reaction using oligonucleo-tides 5�-TTG GTA CAT ACG GTG ACC TTT TAT TGTTTC G-3� (NcoI) and 5�-CCT GAA CAA GAA CCA TGGGCT GTG ACC-3� (BstEII). These two restriction enzymesites were used to subclone CD36 into the baculoviral trans-fer vector BlueBac4.5 (Invitrogen) containing a 12-histidinetag (36) to generate BlueBac-CD36–12His. The transfer vec-tor was used to engineer a recombinant baculovirus usingthe Bac-N-Blue system (Invitrogen) as directed. For expres-sion in mammalian cells, the modified CD36 cDNA was sub-cloned, replacing the rodent sequence in pCI-Cd36–12His(36) using the restriction enzymes NcoI and BstEII to gener-ate pCI-CD36–12His.

Site-directed Mutagenesis of CD36—Site-directed mutagen-esis was performed on pCI-CD36–12His using theQuikChange multisite mutagenesis kit (Stratagene) accordingto the manufacturer’s instructions. Primers were designed toreplace the asparagine codon in each putative glycosylation sitewith a codon for glutamine. The entire coding sequence of eachmutant was confirmed by DNA sequencing. The sequences ofmutagenic primers for the asparagine to glutamine mutantswere: N79Q, 5�-CAC AGG AAG TGA TGA TGC AGA GCTCCA ACA TTC AAG TTA AGC-3�; N102Q, 5�-TCG TTTTCTAGCCAAAGAACAGGTAACCCAGGACGCTG-3�;N134Q, 5�-TGG AAC AGA GGC TGA TCA GTT CAC AGTTCT CAA TC-3�; N163Q, 5�-GTT CAA ATG ATC CTC AATTCA TTA ATT CAG AAG TCA AAA TCT TCT ATG TTCC-3�; N205Q, 5�-GGT CTG TTT TAT CCT TAC CAG AATACG GCA GAT GGA GTT TAT AAA G-3�; N220Q, 5�-GTTTTC AAT GGA AAA GAT CAG ATC TCT AAA GTT GCCATA ATC G-3�; N235Q, 5�-CAT ATA AAG GTA AAA GGCAGC TGT CCT ATT GGG AAA G-3�; N247Q, 5�-CAC TGCGAC ATG ATT CAG GGT ACA GAT GCA GCC-3�; N321Q,5�-GAAAAAATTATCTCAAAGCAATGTACATCATATGGT GTG CTA G-3�; N417Q, 5�-TGT GCC TAT TCT TTGGCT CCA GGA GAC TGG GAC CAT TGG-3�.Mammalian Cell Culture and Protein Expression—

HEK293T cells were grown in Dulbecco’s modified Eagle’smedium supplemented with 10% fetal bovine serum (Invitro-gen) at 37 °C in 5% CO2. The cells were transfected transientlywith wild type and mutant pCI-CD36–12His or pCI-ABCB1–12His, as described previously (37). 24 h after transfection thecells were treated with 33 �M butyric acid (Sigma) and culturedfor 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-transfectionwhere indicated.Insect Cell Culture and Protein Expression—Suspension

cultures of Spodoptera frugiperda 21 (Sf21) cells were grownin SF900II serum-free medium (Invitrogen) at 27 °C withshaking at 100 rpm. Cells at a density of 2 � 106 cells/ml wereinfected with recombinant baculovirus encoding wild typeor non-glycosylated CD36 using a multiplicity of infection ofat least 3 viruses per cell. After several hours the culture wasdiluted to a density of 1 � 106 cells/ml with fresh SF900IImedia.Insect Cell Membrane Preparation—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, pH7.5, 250mMsucrose, 0.2mMCaCl2, 2mMbenzamidine,40 �M leupeptin, and 1 �M pepstatin A). The cells were resus-pended in 10ml of buffer 1 and frozen at�20 °C. Once thawed,the cells were homogenized at 4 °C by 5 � 30-s bursts at 24,000rpm (DI 25 homogenizer; Yellow Line). The sample was centri-fuged at 500 � g for 10 min at 4 °C to pellet the large organellesand unbroken cells. The supernatant was recovered and centri-fuged at 100,000� g in aTLA100.3 rotor (BeckmanCoulter) for50 min at 4 °C to obtain pelleted membranes. The crude mem-brane fraction was resuspended in buffer 2 (buffer 1, minusCaCl2) supplemented with 10% (v/v) glycerol and stored at

Role of Glycosylation in CD36

16278 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 • JUNE 12, 2009

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

�80 °C. Total protein concentrations of the membrane frac-tions were determined by DC Protein Assay (Bio-Rad).Solubilization and Purification of CD36 from Membrane

Fractions—Membrane fractions were pelleted by centrifuga-tion at 100,000 � g for 30 min in a TLA100.3 rotor at 4 °C. Thepellets of wild type CD36 were resuspended in solubilizationbuffer (20mMTris-HCl, pH6.8, 2% (w/v)OG, 150mMNaCl, 1.5mMMgCl2, 5% (v/v) glycerol, 2mMbenzamidine, 40�M leupep-tin, and 1 �M pepstatin A) at 5 mg protein/ml, homogenized byextrusion in a 21-gauge needle, and constantlymixed for 90minat 4 °C. The insoluble fraction was pelleted by ultracentrifuga-tion at 100,000 � g for 30 min in a TLA100.3 rotor at 4 °C.Ni-NTA resin was pre-equilibrated in equilibration buffer (sol-ubilization bufferwhere 2%OGwas replacedwith 1%OG in thepresence of 20 mM imidazole). Imidazole (20 mM) was added tothe solubilized fraction of wild type CD36 membranes andincubated with the Ni-NTA resin using a protein:resin ratio of8:1 with continuous mixing for 1 h at 4 °C. The resin waswashed 4 times with 20 bed volumes and a stepwise gradient ofimidazole (60–120mM) inwash buffer (20mMTris-HCl pH8.0,150mMNaCl, 1.5mMMgCl2, 5% (w/v) glycerol, 1% (w/v) OG, 2mM benzamidine, 40 �M leupeptin, and 1 �M pepstatin A) toeliminate proteins bound non-specifically to the resin. Wildtype CD36 was eluted using equilibration buffer plus 250 mMimidazole. The purification efficiency was visualized by SDS-PAGE stained with colloidal blue. An identical procedure wasused for purification of non-glycosylated CD36 (CD36non-g)except 0.6% SDS substituted 2%OG in the solubilization bufferand 0.3% SDS replaced 1% OG in the equilibration and washsolutions. The eluted protein was concentrated using centrifu-gal devices with a 50-kDa cut off as directed (Amicon Ultra 15,Millipore).Deglycosylation of PurifiedWild TypeCD36 Protein—For use

in mass spectrometry, �10 pmol of wild type CD36 was dena-tured at 100 °C for 10 min and deglycosylated using PNGase Ffor 1 h at 37 °C as directed (New England Biolabs).Mass Spectrometry—Approximately 10 pmol of purified wild

type CD36 (pre and post-deglycosylation) were separated bySDS-PAGE and stained with colloidal blue. The protein bandswere excised and digestedwith trypsin usingMassPREP Station(Waters) for the liquid chromatography/tandem mass spec-troscopy (LC/MS/MS) or BioRobot 3000 (Qiagen) for the Fou-rier transform ion cyclotron resonance (FT-ICR MS). Peptideswere extracted using 0.1% formic acid, and the tryptic peptidemixture was analyzed by automated LC/MS/MS (CapLC, LCPackings, Q-ToF II, Waters) as described (38) or Fourier trans-form mass spectrometry (LTQ-FT hybrid linear trap/7-T FT-ICRmass spectrometer (Thermo Electron, Bremen, Germany))as described (39). Data from LC/MS/MS and FT-ICRMS wereanalyzed in conjunction with the MSDB data base using thesoftware tool Mascot (Matrix Services) and Sequest data baseusing Bioworks software (Thermo Scientific), respectively.Flow Cytometry—After harvest, transiently transfected

HEK293T cells were washed in FACS buffer (PBS and 1% fattyacid-free BSA) and resuspended at 1 � 107 cells per ml. A sat-urating concentration (2�g) ofmouse anti-CD36mAb1258 (ormAb1955) was added to 50 �l of cells and incubated for 30minat 4 °C (0.5 �g of monoclonal antibody 4E3 (DAKO) was sub-

stituted when staining for P-glycoprotein). The cells were cen-trifuged at 400 � g for 1 min at 4 °C and resuspended in 1 ml ofFACS buffer. The wash step was repeated twice more, and thecells were resuspended in 50 �l of FACS buffer. A saturatingconcentration (4 �g) of goat, anti-mouse IgG secondary anti-body conjugated to R-phycoerythrin (DAKO) was added to thecells and incubated in the dark for 30min at 4 °C. The cells wererecovered by centrifugation and washed as before and thenresuspended in 400 �l of FACS buffer. During flow cytometry,10,000 cells of normal size and granularity were analyzed forCD36–12His surface expression measuring R-phycoerythrinfluorescence (Ex 565 nm and Em 578 nm). The cell surfaceexpression of CD36–12His was analyzed by FlowJo (Treestar).The cells typically exhibited a biphasic staining pattern, likelydependent on whether the individual cell was just about to, orhad recently divided, before transfection. The heights of thesepeaks (reflecting cell number) relative to each other sometimesvaried, but the fluorescence intensity of each peak (reflectingCD36 density) remained consistent relative to the positive andnegative controls (wild type CD36 and CD36non-g, respec-tively). The maximal expression level of the receptor (the peakwith the higher fluorescence intensity) was, therefore, gated,and the median was calculated and compared in all experi-ments. The expression level of mutant CD36 proteins wasalways compared with the expression levels of wild type andnon-g proteins performed contemporaneously to control forminor variability in transfection efficiency on different days.-Fold reduction in expression was calculated by dividing thepercentage surface expression of the wild type protein by thepercent surface expression of the mutant. The synergy factorwas calculated by dividing the -fold reduction of the multiplemutant by the product of the -fold reduction of the individualmutants, as described (40).In Vitro Solid Phase Ligand Binding Assay—For purified pro-

tein, 1 �g of wild type CD36–12His was added to Ni-NTA-coated plates (Qiagen) in 100 �l of protein binding buffer (20mM 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 �M leupeptin,and 2 �M pepstatin A) and bound at 4 °C overnight with gentlerocking. Unbound protein was removed, and each well waswashed with 2 � 150 �l of ligand binding buffer 1 (LBB1; PBS,1 mM MgCl2, 1 mM CaCl2, 0.5% (w/v) OG, 0.2% fatty acid-freeBSA). Increasing concentrations of BODIPY Ac-LDL wereadded in LBB1 to a total volume of 100 �l and incubated atroom temperature in the dark with gentle rocking for 2 h.Unbound ligand was removed, and the wells were washed with3 � 200 �l of ice-cold wash buffer 1 (PBS, 1 mM MgCl2, 1 mMCaCl2, 0.5% fatty acid-free BSA). 100 �l of PBS was added perwell before determining the bound fluorescence using a fluo-rescent plate reader (SpectraMax, Gemini EM, MolecularDevices, Ex 485 nm, Em 530 nm). Specific binding of Ac-LDLwas calculated after subtraction of the level of BODIPYAc-LDLbound non-specifically to empty wells.Cell-based Ligand Binding Assay—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, asdescribed above. 48 h after transfection, the cells were washedwith 3 � 200 �l wash buffer 2 (WB2; PBS, 1 mM MgCl2, 1 mM

Role of Glycosylation in CD36

JUNE 12, 2009 • VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16279

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

CaCl2), then 150�l of block buffer (WB2with 1% fatty acid-freeBSA) was added to each well and incubated at room tempera-ture for 45 min with gentle rocking. Increasing concentrationsof BODIPY Ac-LDL were added in WB2 plus 0.2% fatty acid-free BSA to a total volume of 100�l and incubated at 4 °C in thedark with gentle rocking for 2 h. Unbound ligand was removed,and thewells werewashedwith 3� 200�l of ice-coldWB2. 100�l of PBS was added per well before determining the boundfluorescence as described above and calculation of the specificbinding by subtraction of the level of BODIPY Ac-LDL boundnon-specifically to mock-transfected cells.LigandBindingAnalysis—Data were analyzed using Graph-

pad Prism software Version 4.0, and saturation binding datawere best fitted by Langmuir adsorption isotherm (Equation1), which describes binding of ligand to a single class of bind-ing site as

B �Bmax � �L�

Kd � �L�(Eq. 1)

where B is bound ligand (relative fluorescent units), [L] is con-centration of ligand (�g/ml), and Kd is the concentration ofligand giving half-maximal binding and ameasure of the affinityof ligand-receptor interaction.Immunoblotting—HEK293T cell lysates (50 �g) (untreated

or treated with 1 unit of Endo H or 1 unit of PNGase F asdirected (New England Biolabs)) were separated by SDS-PAGEand transferred to polyvinylidene difluoride membranes (Mil-lipore).Western blots were probed with rat anti-CD36 primarymAb1955 (or mouse anti-P-glycoprotein primary C219) andrabbit anti-rat (or goat anti-mouse as appropriate) secondaryantibody-conjugated to horseradish peroxidase (DAKO) beforevisualization by ECL chemiluminescent detection system(Amersham Biosciences) as directed.

RESULTS

Overexpression, Purification, and Functional Integrity ofHuman CD36 in Insect Cells—Using the consensus sequenceN-X-Ser/Thr (whereX is any amino acid except proline), aspar-agines 79, 102, 134, 163, 205, 220, 235, 247, 321 and 417 (hence-forth designated N1—N10 for convenience) were identified aspossible sites of modification. Mass spectrometric analysis ofpurified protein can be used to determine the occupancy ofthese sites; therefore, recombinant wild type CD36 was engi-neered with a 12 histidine, carboxyl-terminal tag and expressedin Sf21 insect cells using a baculoviral system. Proteins weresolubilized in OG from insect cell membrane fractions, and therecombinant CD36 was purified by nickel-NTA affinity chro-matography (Fig. 1A). Although insect cells faithfully recognizethe mammalian glycosylation sites (41), they do not elaborateon the core glycan paucimannose (42). It was, therefore, impor-tant to establish that the CD36 produced in the heterologoushost was functional and, thus, suitable for analysis. The purifiedprotein was immobilized on plates coated with Ni-NTA and itsaffinity for Ac-LDL was determined (Fig. 2A; Table 1). Themean Kd � S.E. � 6.4 � 1.5 �g/ml (n � 3) for Ac-LDL of thereceptor purified from insect cells was not significantly differ-ent from the affinity measured for the recombinant protein

expressed on the surface of mammalian cells (Kd � 8.3 � 1.4�g/ml (n � 4); Fig. 2B; Table 1). Thus, the purified proteinretains its native fold and ligand binding function, and the spe-cific nature of the glycan is not important for ligand interaction.Identification of N-Linked Glycosylation Site Occupancy of

CD36 by Mass Spectrometry, Mutagenesis, and ElectrophoreticMobility—Digestion of theCD36 purified from insect cells withPNGase F increased the mobility of the protein during SDS-PAGE (Fig. 1B). The apparent mass difference of 20 kDa sug-gests several of the 10 putative glycosylation sites are modified.The PNGase F-treated CD36 migrated with similar mobility toa non-glycosylatable version of the protein (CD36non-g,described below) consistent with completeN-linked deglycosy-lation of wild type CD36. Deglycosylation of a protein byPNGase F removes theN-linked oligosaccharide from the pep-tide chain and converts the previously glycosylated asparagineto an aspartic acid. This results in a mass shift of 1 for eachdeamidation event as compared with the predicted molecularmass of the protein backbone containing a non-utilized aspar-agine (the molecular mass of a glycopeptide is difficult to pre-dict and to measure because of the heterogeneity of the sugarchains and increased mass to charge ratio). We used this massshift associated with the deglycosylation to assess the glycosy-lation status of the protein by Q-ToF mass spectrometry aftertryptic digestion. Complete tryptic digest of wild type CD36generates 46 fragments, of which 7 contain the putativeN-linked glycosylation sites. InQ-ToFMS, peptides are ionizedto form, among others, different y-series carboxyl-terminal andb-series amino-terminal ions, and analysis of the mass and

FIGURE 1. Purification of CD36 –12His by affinity chromatography anddeglycosylation by PNGase F. A, SDS-polyacrylamide gel stained with col-loidal blue showing protein fractions during the purification of wild typeCD36 –12His from baculoviral-infected Sf21 cells. Lane 1, 50 �g of crude mem-brane fraction (0.1% of starting material); lane 2, OG-solubilized membraneproteins (0.1% by volume); lane 3, proteins failing to bind to the Ni-NTA (0.1%by volume); lanes 4 – 8, washes 60 –120 mM imidazole of the Ni-NTA resin toremove proteins non-specifically bound (2% by volume); lanes 9 –12, elutionfractions in 250 mM imidazole (2% by volume). B, the electrophoretic mobilityof purified recombinant wild type CD36 (lane 1) was compared with CD36after deglycosylation by PNGase F (lane 2) and non-glycosylatableCD36non-g purified from protein aggregates (lane 3).

Role of Glycosylation in CD36

16280 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 • JUNE 12, 2009

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

charge of these ions determines the amino acid sequence of thepeptide (Fig. 3A).For example, tryptic fragment 235NLSYWESHCDMINGT-

DAASFPPFVEK260 contains 2 putative glycosylation sites, N7

and N8, with N7 at the amino terminus of the fragment. As N7is at the amino terminus, the b-ions determine whether N7 hasbeen deglycosylated and deamidated to an aspartic acid (Fig.3B, upper panel). The b-ions with m/z 229.12 (b2) and m/z316.15 (b3) are shown on the spectrum, and both are diagnosticfor aspartic acid at position 235, indicative of modification ofthe wild type Asn (N7) by glycosylation. Putative glycosylationsite N8 is located in the middle of the tryptic fragment; there-fore both b-ions and y-ions can determine whether this site isoccupied. The b-ion with m/z 1709.66 (b14) is diagnostic foraspartic acids in both positions 235 and 247, and the y-ionswithm/z 1593.78 (y15) and 1480.70 (y14) indicate an aspartic acid inposition 247, indicative that N8 is glycosylated in wild typeCD36 (Fig. 3B, lower panel). Fragments with asparagines thatare not within consensus glycosylation sites show little evi-dence of similar mass change, indicating that spontaneousdeamidation is rare (data not shown).Q-ToFMS on tryptic digest fragments fromwild type CD36,

in the absence of PNGase F treatment, failed to detect any frag-ments containing putative glycosylation sites, although otherfragments of CD36, not predicted to be glycosylated, weredetected. This too, is consistent with glycosylation of the rele-vant fragment, as the sensitivity of glycopeptide detection isoften low due to signal suppression and the likely heterogeneityof glycosylation. Q-ToF mass spectrometry of the deglycosy-lated protein identified five of the seven tryptic fragmentssought and was able to establish that N1, N5, N7, N8, N9, andN10 were utilized as glycosylation sites (full spectra, data tablesof the ions detected, and their mass errors from the calculatedvalues are shown in supplemental Fig. 1, A–E).

The remaining four putative glycosylation sites were locatedin tryptic digest fragments too large (6953 mass units for N2,N3, N4) or too small (577 mass units for N6) to be detected byQ-ToF MS. We, therefore, utilized FT-ICR MS to attempt todetermine the glycosylation status of these putative sites. FT-ICR MS achieved 42% coverage of the protein and identifiedthat N6 can also be glycosylated (Fig. 3C shows the spectrumfrom a partial tryptic digest product containing N6). However,not all fragments exhibited this mass shift, indicating that N6modification is unlikely to be comprehensive (the full spectra ofboth these fragments are recorded in supplemental Fig. 1, F andG). Again, the fragment containing N2, N3, and N4 was notdetected. This is most likely due to a low charge state for thepeptide, which has only one internal histidine, precluding anm/z value lower than 2000. As digestion with other proteasesdid not result in fragments suitable for detection by mass spec-trometry, electrophoretic mobility of CD36 isoforms with andwithout the test site was used to determine the glycosylation

FIGURE 2. The affinity of CD36 for ligand is not affected directly by thenature or pattern of glycosylation. Interaction of CD36 proteins with Ac-LDL was measured using increasing concentrations of BODIPY Ac-LDL tocore-glycosylated, wild type (wt) CD36 purified from Sf21 insect cells andimmobilized on a solid phase (A), mature glycosylated, wild type CD36 on thesurface of HEK293T cells (B), and CD36N8 –10 on the surface of HEK293T cells(C). A representative graph from each is shown and is plotted according toEquation 1; the Kd quoted represents the mean � S.E. (�g/ml) from at leastthree independent experiments.

TABLE 1Binding affinity of BODIPY Ac-LDL to CD36 proteins

Protein Kd (mean � S.E.)�g/ml

Wild type CD36 (purified from Sf21) 6.35 � 1.47 n � 3Wild type CD36 (expressed in HEK293T) 8.26 � 1.38 n � 4CD36N8–10 (expressed in HEK293T) 5.73 � 1.54 n � 4CD36N1–7,9,10 (expressed in HEK293T) 6.21 � 0.03 n � 2CD36N1–8,10 (expressed in HEK293T) 4.12 � 0.32 n � 2CD36N1–9 (expressed in HEK293T) 4.24 � 0.03 n � 2

Role of Glycosylation in CD36

JUNE 12, 2009 • VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16281

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

FIGURE 3. Mass spectrometry showing evidence of glycosylation of sites N6, N7, and N8 of CD36 purified from Sf21 insect cells. After tryptic digestion,protein fragments are ionized into related y- and b-series protonated ions (as shown by the schematic in A) with unique mass/charge (m/z) detectable by MS.B, selected portions of Q-ToF MS spectra of the PNGase F and trypsin-digested wild type CD36. The spectra show the diagnostic ions, which demonstrate thatN7 and N8 are deglycosylated and deamidated to aspartic acids by treatment with PNGase F, result in a mass shift of 1 each. C, selected portion of FT-ICR MSspectrum of similarly treated wild type CD36 showing that N6 can be glycosylated. The sequence of the tryptic digest fragments with the putative glycosylatedasparagine underlined and the complete y and b-series ions generated by the ionization of the PNGase-F-deglycosylated and -deamidated fragment areshown. For Q-ToF MS, those single-charged ions detected by MS are shown in bold. Fragments not detected are shown in italics. Note in the FT-ICR MSspectrum, some of the y- and b-series ions have more than one charge. The sequence of the fragments and the observed and expected Mr of the monoisotopicparent ion (accounting for the deamidation of the underlined Asn to Asp) are given above the spectra. For the full spectra, tables of ions detected and deviationsfrom the expected ion mass, see supplemental Fig. 1.

Role of Glycosylation in CD36

16282 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 • JUNE 12, 2009

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

status of these putative sites and corroboration of the incom-plete utilization of N6.Glycosylation Status of Sites N2, N 3, N4, andN6—Reasoning

that the difference in electrophoretic mobility would be moreevident in a less complex CD36 template with fewer glycosyla-tion sites, we first established that CD36N8–10, with only thecarboxyl-terminal three sites intact, trafficked to the cell sur-face (see below for characterization of this mutant).CD36N8–10 migrated with an apparent molecular mass of�55 kDa on SDS-PAGE (Fig. 4A). A series of mutants weregenerated based on the CD36N8–10 template to test the utili-zation of putative glycosylation sites N2, N3, N4, andN6. Thesesites were reintroduced individually into the minimally glyco-sylated template to generate CD36N2,8–10, CD36N3,8–10,CD36N4,8–10, and CD36N6,8–10 (Table 2). These mutants,together with CD36N8–10, were expressed in HEK293T cells,and the electrophoretic mobility of the proteins was comparedby SDS-PAGE and immunoblotting by probing withmAb1955.It was hypothesized that if the introduced putative glycosyla-tion site is occupied in any of the mutants, the electrophoreticmobility of this protein is likely to be slower than that ofCD36N8–10. As a positive control, CD36N7–10 (N7 wasshownbyQ-ToFMS to be glycosylated in thewild type protein)was compared with CD36N8–10 and found to migrate moreslowly through the gel (Fig. 4A, upper panel). The immunoblotsuggests that N2, N3, and N6 are glycosylated as CD36N2,8–10, CD36N3,8–10, and CD36N6,8–10 migrate more slowlythan CD36N8–10. After treatment with PNGase F, all of theCD36 proteins migrate with the same mobility (Fig. 4A, lowerpanel), confirming that the glycans on N2, N3, and N6 wereresponsible for the electrophoretic difference observed in Fig.4A. In repeated experiments, inclusion of site N6 in theCD36N8–10 consistently resulted in partial glycosylation ofthe protein as judged by the significant level of multiple proteinforms in lane 6 of the upper panel of Fig. 4A, which are resolvedinto a single species in the lower panel by treatment with

PNGase F. Although we cannot rule out the possibility thatinclusion of N6 may influence the occupancy of sites N8–N10,the simplest interpretation, and one that is consistent with theFT-ICR data on the wild type protein, is that N6 itself is recog-nized inefficiently as a glycosylation site. In these experimentswe found no evidence to suggest N4 is modified by glycosyla-tion. Taken together with the mass spectrometry data, it is evi-dent that 9 of the 10 putative glycosylation sites can bemodifiedby the glycosyl transferases.Glycosylation Is Necessary for Trafficking of CD36 to the Cell

Membrane—Toassess the role of glycosylation in the biology ofCD36, we first removed all 10 putative glycosylation sites,replacing the conserved asparagines with glutamines by site-directed mutagenesis to generate CD36non-g. The mutantcDNA was expressed in the human cell line HEK293T.CD36non-g is translated by HEK293T cells as shown byWest-ern blotting, probing with the anti-CD36 monoclonalmAb1955 (Fig. 5A), but when intact cells were analyzed by flowcytometry for surface expression usingmAb1258 (ormAb1955,data not shown), it is clear that CD36non-g fails to traffic to theplasma membrane (Fig. 5B).Determination of theMinimal Glycosylation to Support Traf-

ficking of CD36—To determine which N-linked glycosylationsites are required for CD36 to reach the cell surface, we reintro-duced glycosylation sites back intoCD36non-g (for details of allmutants generated, see Table 2). Flow cytometry was used toanalyze the level of cell surface expression of the mutated pro-teins expressed in HEK293T cells. Saturating levels (data notshown) of primary mAb1258 and secondary antibodies wereused to allow quantification of surface expression. FACS anal-ysis of twomutants with an overlapping pattern of intact glyco-sylation sites N1–7 (CD36N1–7) or N7–10 (CD36N7–10) (Fig.6A) showed that both reached the plasma membrane but wereexpressed to very different levels (1.6 and 11% of wild typeCD36 (wtCD36), respectively; the top section of Table 3)(althoughonly 1.6%of the level ofwild type, antibody binding tocells expressing CD36N1–7 is significant, being 20-fold greater

FIGURE 4. Electrophoretic mobility shift analysis to test glycosylation statusof CD36. A, whole cell lysates (50 �g of protein) from transiently transfectedHEK293T cells were analyzed by immunoblotting probing with mAb1955 before(upper panel) and after (lower panel) deglycosylation with PNGase F. Lane 1,CD36N8–10; lane 2, CD36N8–10N7; lane 3, CD36N8–10; lane 4, CD36N8–10N3; lane 5, CD36N8–10N4; lane 6, CD36N8–10N6; lane 7, CD36N8–10;lane 8, CD36N8–10N7; lane 9, CD36N8–10N2. B and C, analysis ofCD36N8–10 probed with mAb1955 (B) and P-glycoprotein probed withmAbC219 (C) after tunicamycin treatment of the transfected cells to inhibit gly-cosylation: lane 1, untreated sample; lane 2, treated with PNGase F; lane 3, treatedwith tunicamycin; lane 4, treated with tunicamycin and PNGase F.

TABLE 2CD36 mutants generated, and details of asparagines that have beenmutated to glutamines

Role of Glycosylation in CD36

JUNE 12, 2009 • VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16283

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

than the background binding associated with CD36non-g).This indicated two things; first, it was likely that there was bothan amino-terminal and a carboxyl-terminal set of glycosylationsites that supported trafficking (unless N7 proved to be the keysite); second, the low level of expression of both mutants sug-gested that sites in both the amino- and carboxyl-terminal setswere likely to be necessary to achieve high level expression ofthe receptor.Characterization of the Amino-terminal Glycosylation Set—

To further assess which of the seven amino-terminal glycosyla-tion sites N1–7 of CD36 (six of which are modified byN-linkedglycosylation) were necessary for the protein to reach the cellmembrane, sites were removed systematically from each end(the second panel of Table 3). The expression levelof CD36N1–6 was essentially the same as CD36N1–7, butCD36N1–5 failed to traffic to the cell surface, whereasCD36N2–7 caused a 5-fold reduction in trafficking (as com-pared with CD36N1–7), and CD36N3–7 failed to reach theplasma membrane (all of the mutants generated were analyzedbyWestern blotting using the monoclonal antibody mAb1955,which recognizes both the wild type and the non-glycosylatedversion of the protein to confirm that the glycosylation sitemutants were synthesized by the HEK293T cells irrespective ofsurface representation (data not shown)). These data could sug-gest the importance of N1, N2, and N6 in the amino-terminalset; however, a mutant with only sites N1, N2, N6, and N7present failed to traffic to the cell surface, indicating that thesesites can only exert an effect in the context of a more heavilyglycosylated protein. Indeed, given that N4 does not appear tobe modified by N-linked glycosylation, there is a correlationbetween glycosylation site density and trafficking within theamino-terminal set. Thus, all mutants with five or more of theutilized amino-terminal glycosylation sites irrespective of com-bination appear on the cell surface, whereasmutantswith fewerthan five utilized sites are unable to support trafficking.This interdependence of glycosylation sites made assessing the

contribution of the amino-terminal sites to the trafficking effi-ciency difficult; however, their influence on the trafficking whencombinedwith thecarboxyl-terminal set suggested that sites2and5 were particularly important (see below). Nevertheless, the dem-onstration that CD36N1–6 can be expressed at the cell surface

and the knowledge that it shares nocommon glycosylation site withCD36N7–10 indicate thatnoneof theglycosylation sites is individuallyessential forCD36 to traffic to the cellsurface.Characterization of the Carboxyl-

terminal Glycosylation Set—Genera-tion and analysis of four mutantsCD36N7,9,10, CD36N8–10, CD36N7,8,10, and CD36N7–9 (third panel ofTable 3) confirmed that N7 was notrequired for traffickingofCD36 to theplasma membrane, because the sur-face expression of CD36N7–10 wasessentially the same as CD36N8-10 (Fig. 6B). The failure of

CD36N7,9,10, CD36N7,8,10, and CD36N7–9 to express atthe cell surface suggested that sites N8, N9, and N10 (all ofwhich were shown by Q-ToF MS to be modified by N-linkedglycosylation) were essential for expression and that the threeintact glycosylation sites in CD36N8–10 were the minimalnumber in the carboxyl terminus that would allow cell surfaceexpression. Indeed, mutant proteins with only two of these gly-cosylation sites, CD36N8,9, CD36N8,10 and CD36N9,10 (thirdpanel of Table 3), were unable to traffic to the cell membrane(Fig. 6C).Carboxyl-terminal Glycosylation Sites Contribute Signifi-

cantly to the Cell Surface Expression in the Presence of the Ami-no-terminal Set—Although the paired carboxyl-terminal gly-cosylation sites were unable to support trafficking of anotherwise non-glycosylatedCD36, the individual sites do have astrong influence on the level of trafficking when amino-termi-nal glycosylation sites are present. This became evident wheneach of the three sites was restored individually to CD36N1–7.CD36N1–7,8, CD36N1–7,9, and CD36N1–7,10 trafficked tothe cell membrane with 34, 30, and 24% efficiency of the wildtype protein (Table 3, fourth panel).Generation of the reciprocal set of mutants (removing each

of the carboxyl-terminal three sites individually from an other-wise wild type CD36) allowed us to assess whether these threesites functioned additively or synergistically to influence thetrafficking of the protein. CD36N1–7,9,10, CD36N1–8,10, andCD36N1–9, trafficked to the cell membrane at, on average, 63,78, and 75% that of the level of wild type CD36 (Fig. 6F; fifthpanel of Table 3). When compared with the trafficking effi-ciency of CD36N1–7, from which all three carboxyl-terminalsites are absent and which is present at the cell surface at 1%that of the level of the wild type protein, there is a strong sug-gestion that the three carboxyl-terminal sites function synergis-tically. This is because the -fold reduction of the individualmutants compared with the wild type range between 1.3 and1.6, but the surface expression of the triple mutant is reduced62.5-fold. The synergy factor, i.e. the -fold reduction of the tri-ple mutant, divided by the product of the -fold reduction of thethree individual mutants, should approximate 1 if the mutantsare additive. Here the synergy factor of 23.1 suggests a strong

FIGURE 5. Non-glycosylatable CD36non-g fails to traffic to the plasma membrane. A, immunoblot analysisprobing with mAb1955 confirms CD36non-g is translated when expressed transiently in HEK293T cells; thelower molecular weight species in the non-g lane is not always observed and is likely to be a degradationproduct. B, whole cell flow cytometry analysis using an antibody (mAb1258) that recognizes an epitope in theextracellular loop of CD36 indicates that CD36non-g fails to traffic to the plasma membrane. Wild type CD36 isshown in black, CD36non-g shown in blue, and untransfected cells shown in red. PE, R-Phycoerythrin.

Role of Glycosylation in CD36

16284 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 • JUNE 12, 2009

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

synergistic linkage between the three sites and trafficking of theprotein (Table 3).Glycosylated Sites in the Amino Terminus Contribute Signif-

icantly to the Cell Surface Expression in the Presence of the Car-boxyl-terminal Set—To assess the contribution of amino-ter-minal glycosylation sites, we restored each, individually, to

CD36N8–10 and assessed their ability to traffic to the plasmamembrane (bottom panel of Table 3). Only site N7 and site N4had non-significant effects on the trafficking of the protein,entirely consistent with our earlier observations that N7 had noeffect on trafficking when part of the amino-terminal set andthat N4 is not utilized as a glycosylation site. Each of the otheramino-terminal sites significantly improve the trafficking ofCD36N8–10, with sites N2 and N5 having the greatest effect,achieving �60% of the cell surface expression level of the wildtype protein (to illustrate, the surface expression ofCD36N2,8–10 is shown in Fig. 6G).Glycosylation, andNot the Reintroduction of the Asparagines,

Determines Trafficking Efficiency—The data indicate the pres-ence of at least twominimal sets of glycosylation sites that sup-port trafficking of CD36 proteins. To rule out the possibilitythat it is the reintroduced asparagines that determines traffick-ing rather than their modification by glycosylation, we usedtunicamycin (an inhibitor of glycan precursor synthesis) to pre-vent modification of the asparagines. Western analysis showedthat glycosylation of the CD36N8–10mutant (Fig. 4B) and alsoof the multidrug resistance P-glycoprotein (Fig. 4C), which cantraffic and function in the absence of glycosylation (43), wasefficiently, albeit incompletely, inhibited in the tunicamycin-treated cells. The ability of these proteins to reach the cell sur-face in tunicamycin-treated and untreated cells was then com-pared by flow cytometry (Fig. 6, D and E). CD36N8–10 in cells

FIGURE 6. Cell surface expression of CD36 mutants. Wild type and mutatedCD36 and P-glycoprotein were expressed transiently in HEK293T cells, andflow cytometry was used to measure the cell surface expression using satu-rating amounts of antibody. Wild type CD36, which traffics efficiently, wasused as the positive control (black trace, where shown), and CD36non-g,which fails to traffic, was used as the negative control (blue trace, whereshown) for all CD36 experiments. PE, R-Phycoerythrin. A, cell surface expres-sion of CD36N7–10 (green) and CD36N1–7 (red). B, CD36N7–10 (green) andCD36N8 –10 (black). C, CD36N8,9 (green), CD36N8,10 (light blue),and CD36N9,10 (red). D, surface expression of CD36N8 –10 in untreated (red)and tunicamycin (tun)-treated cells (green). E, surface expression of P-glyco-protein (Pgp) in untreated (red) and tunicamycin-treated cells (green). F, cellsurface expression of CD36N1– 8,10 (red), CD36N1–9 (green), and CD36N1–7,9,10 (light blue). G, cell surface expression of CD36N2,8 –10.

TABLE 3The cell surface expression of mutant CD36 compared to wild type,measured by flow cytometrySurface expression values are calculated for the cell subpopulations with the highestfluorescence and normalized to 100% for the wild type protein and 0% forCD36non-g (i.e. the same fluorescence as background binding to mock-transfectedcells).

CD36 mutant Surface expression % � S.D. (n � 3)CD36 wild-type 100CD36non-g 0CD36N1–7 1.6 � 0.3CD36N7–10 11 � 1.7CD36N1–7 1.6 � 0.3CD36N1–6 1.3 � 0.6CD36N1–5 0CD36N2–7 0.3 � 0.2CD36N3–7 0CD36N1,2,6,7 0CD36N7–10a 11 � 1.7CD36N8–10 12 � 2.0CD36N7,9,10 0CD36N7,8,10 0CD36N7–9 0CD36N8,9 0CD36N8,10 0CD36N9,10 0CD36N1–7,8 34 � 2.0CD36N1–7,9 30 � 1.7CD36N1–7,10 24 � 2.1CD36N1–7,9,10 63 � 14CD36N1–8,10 78 � 5.5CD36N1–9 75 � 4.0CD36N8–10 12 � 2.0CD36N1,8–10 31 � 4.2CD36N2,8–10 59 � 5.6CD36N3,8–10 31 � 3.2CD36N4,8–10 13 � 1.5CD36N5,8–10 60 � 5.0CD36N6,8–10 31 � 3.2CD36N7,8–10a 11 � 1.7

a These are the same mutant.

Role of Glycosylation in CD36

JUNE 12, 2009 • VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16285

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

treated with tunicamycin failed to reach the cell surface (Fig.6D), but P-glycoprotein did (Fig. 6E). Repetition of the experi-ment with the CD36N1–7mutant (data not shown) gave a verysimilar result to CD36N8–10. The simplest interpretation ofthese data is that it is the N-linked glycosylation of CD36 thatenables the protein to fold and traffic correctly.Mature Glycosylation Is Not Necessary for Cell Surface

Expression of CD36—Western analysis of the mutants gener-ated (probed with mAb1955) confirmed that all proteinsincluding those that failed to traffic to the plasma membranewere synthesized by HEK293T cells. Those proteins with uti-lized glycosylation sites that fail to reach the cell surface do,also, become glycosylated, indicative of translation at, andtranslocation into, the endoplasmic reticulum. For example,the electrophoretic mobility of CD36N7,9,10 is increased notonly by digestionwith PNGase F but also by endoglycosidaseH,which cleaves immature, high mannose, glycans from the pro-tein (Fig. 7A). The presence of glycosylation in proteins that donot appear on the cell surface indicates that the mutant is stuckin the trafficking pathway. A fraction of wild type CD36 alsoappears sensitive to endoglycosidase H (judging by the slightincrease inmobility of themain protein band in Fig. 7A, lane 2);however, whether high mannose glycans occupy the same gly-cosylation site in each molecule or whether the slight endogly-

cosidase H sensitivity reflectsincomplete maturation of glycansover the population as a wholeremains to be established.We also analyzed the nature of the

glycan in CD36 isoforms that traf-ficked to thecell surfaceandwere sur-prised to find that a number ofmutants were sensitive to endoglyco-sidase H. For example, CD36N1–7andCD36N8–10,whichsharenogly-cosylation sites in common, are sensi-tive to deglycosylationbyEndoH (Fig.7, B and C, respectively). This doesnot appear to be related to the effi-ciency of trafficking, as CD36N2,8–10, which is expressed on the cellsurface at 59% that of wild type lev-els (Fig. 6G), can be efficiently degly-cosylated by Endo H (Fig. 7D). Onthe other hand, CD36N1–7,9,10,which lacks site N8 but is expressedat the same level as CD36N2,8–10,is substantially more resistant todeglycosylation by Endo H, indicat-ing that its glycans are, predomi-nantly, of a mature form (Fig. 7E).The glycans onCD36N1–7,8,10 andCD36N1–9, each of which is miss-ing one of the three carboxyl-termi-nal sites, also have predominantlymature glycans. However, each ofthe three double site mutants(CD36N1–7,8, CD36N1–7,9, and

CD36N1–7,10) is largely sensitive to endoglycosidase H(CD36N1–7,9 is shown in Fig. 7F to illustrate).Glycosylation of Specific Sites on CD36 Is Not Necessary for

Ligand Binding—Solid phase ligand binding assay was used toestablish whether the minimally glycosylated mutant CD36proteins retained ligand binding functionality. Equilibriumbinding over a range of ligand concentrationswas used tomeas-ure the affinity of CD36N8–10 and CD36N1–7 for Ac-LDL.The Kd values of wild type CD36 and CD36N8–10 are not sig-nificantly different, as determined by unpaired Student’s t test(95% confidence level) (Fig. 2C; Table 1); however, we failedto detect binding to CD36N1–7 (data not shown). The absenceof binding to CD36N1–7 may have meant that N8–10 areessential for binding of Ac-LDL; however, affinity is retained inthe CD36N1–7,9,10, CD36N1–8,10, andCD36N1–9 (Table 1),each of which is missing one of the three carboxyl-terminalsites. It is likely, therefore, that no individual glycan is impor-tant for ligand binding, and the failure to detect binding toCD36N1–7 is due to the very low surface expression level of thismutant.

DISCUSSION

The extracellular domain of the class B scavenger receptorCD36 is heavily modified by glycosylation. Ten consensus gly-

FIGURE 7. Some trafficking-competent CD36 isoforms are only core-glycosylated. 50 �g of whole cellprotein lysates from transiently transfected HEK293T cells treated with either 1 unit of Endo H or PNGase F wereanalyzed by immunoblotting probing with mAb1955. A, wild type CD36: lane 1, untreated; lane 2, treated withEndo H; lane 3, treated with PNGase F. CD36N7,9,10: lane 4, untreated; lane 5, treated with Endo H; lane 6,treated with PNGase F; lane 7, CD36 non-g. B, CD36N1–7: lane 1, untreated; lane 2, treated with Endo H; lane 3,treated with PNGase F. C, CD36N8 –10: lane 1, untreated; lane 2, treated with Endo H; lane 3, treated withPNGase F. D, CD36N2,8 –10: lane 1, untreated; lane 2, treated with Endo H; lane 3, treated with PNGase F.E, CD36N1–7,9,10: lane 1, untreated; lane 2, treated with Endo H; lane 3, treated with PNGase F. F, CD36N1–7,9:lane 1, untreated; lane 2, treated with Endo H; lane 3, treated with PNGase F.

Role of Glycosylation in CD36

16286 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 • JUNE 12, 2009

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

cosylation sites (asparagines 79 (N1), 102 (N2), 134 (N3), 163(N4), 205 (N5), 220 (N6), 235 (N7), 247 (N8), 321 (N9), and 417(N10)) are present in this domain of the human form of theprotein. Having established that the protein produced heter-ologously in insect cells and purified to near homogeneity isfunctionally equivalent to that expressed in mammalian cells,we used mass spectrometry to establish unequivocally thatseven sites (N1, N5, N7, N8, N9, N10) were modified in wildtype CD36. Evidence of glycosylation of N6 was observed insome but not all of the fragments detected. The central trypticdigest fragment containing the other three putative glycosyla-tion sites was not detected by mass spectrometry, and a differ-ent analysis was required to determine the utilization of sitesN2, N3, and N4. Reasoning that a molecular mass differenceassociated with an individual glycosylation event would bemore reliably measured in a less complex glycoprotein with asmaller molecular mass, we first determined that N8, N 9, andN10 were the minimum number of carboxyl-terminal sites ofglycosylation required for trafficking of CD36 to the plasmamembrane.Adding each of the three test sites (N2, N3, N4) individually

to this template and, also N6, because the mass spectrometrysuggested incomplete utilization of this site, produced amodestbut significant reduction in the rate of migration in proteinscontaining N2, N3, or N6. These sites are therefore exposed to,and can be modified by, the glycosyltransferases on transitthrough the folding and trafficking pathway. However, againthe data suggested that N6 is not always modified. No evidencewas found for modification of N4. Thus, 9 of the 10 consensusglycosylation sites in human CD36 are likely to be modified byglycosylation, and of the 7 glycosylation sites conservedbetween human CD36 and bovine Cd36 (in which all 8 arereported to be utilized (34)), all were shown to be glycosylatedin human CD36.Non-glycosylatable CD36, with all 10 consensus sites

mutated, fails to reach the cell surface, indicating that glycosy-lation is necessary for correct trafficking of the protein.CD36non-g protein is pelleted at 100,000 � g from mechani-cally disrupted insect cells along with cellular membranes. Thissuggests that CD36non-g is retained in an intracellular fractionwith a high molecular weight. When combined with the insol-ubility of CD36non-g in all but the strongest ionic detergent(SDS), it seems likely that the protein is shunted from the fold-ing and trafficking pathway to formprotein aggregates, perhapssimilar to bacterial inclusion bodies.The total absence of trafficking of CD36non-g can be rescued

by restoring glycosylation sites to the amino-terminal half(CD36N1–7) or to the carboxyl-terminal half (CD36N8–10) ofthe protein, demonstrating a level of redundancy in the glycanpattern needed for cell surface expression.Neither of these pro-teins is expressed highly, achieving 1.6 and 12% that of the wildtype CD36 level, respectively. However, after restoration of N2or N5 to CD36N8–10, a dramatic increase in expression can beattained (up to 60% that of thewild type level). Similarly, each ofthe carboxyl-terminal three sites is able to improve traffickingof the N1–7mutant by up to 20-fold, suggestive of a synergisticrelationship between the amino- and carboxyl-terminal glyco-sylation events for folding and trafficking of the protein.

CD36N8–10 retained its protein fold, being able to bindligand with the same affinity as the wild type protein. However,none of these three carboxyl-terminal glycanswas necessary forligand binding because mutant proteins devoid of any one ofthe three sites retain affinity for ligand. In contrast, CD36N1–7was not shown to bind ligand. This most likely reflects the lowlevel expression of the N1–7 form of the protein. At only 1% ofthe level of the wild type protein, it may be impossible to dis-tinguish specific binding to the CD36N1–7 in HEK293T cells.We cannot formally rule out the possibility that the carboxyl-terminal sites are specifically required for proper folding of theligand binding domain, but this seems unlikely because all threeof the singlemutants,missing onlyN8 orN9 orN10, retain highaffinity for ligand.A major surprise was the finding that the glycans on these

partially glycosylated proteins remained immature and sensi-tive to deglycosylation by Endo H. This is true of proteins thatshare no sites in common (e.g. CD36N1–7 and CD36N8–10),which together include all nine utilized glycosylation sites in theprotein, some or all of which must be occupied by mature gly-cans in thewild type protein. It is also true of some proteins thatare highly expressed (e.g. CD36N2,8–10). Although it is clearfrom our analysis of the wild type protein sourced from insectcells which are unable to elaborate core glycosylation that thenature of the glycan is not absolutely required for appropriatefolding, trafficking, and ligand binding activity of CD36, it is notclear why the immature glycans on CD36N2,8–10 fail tobecome matured in the Golgi compartment of the mammaliancells. However, although our data are not conclusive in thismatter, there is a correlation between site density on the proteinandmaturation of the glycans. The glycans on, for example, thesingle site mutants devoid of either N8, N9, or N10 (each ofwhich contains eight glycosylation sites) become matured, yetthose on the three double mutants CD36N1–7,8, CD36N1–7,9and CD36N1–7,10 (each with seven glycosylation sites),CD36N1–7 (six glycosylation sites), or CD36N2,8–10 (fourglycosylation sites) remain immature.Our data are most consistent with a role for glycosylation in

the folding and trafficking of the receptor. Potentially, properfolding of the protein could have been due to induction byintramolecular interaction of the amino acid side chains withthe added glycans, as hypothesized in the folding of Erythrinacorallodendron lectin (31, 44). However, althoughCD36N8–10and CD36N1–7 do not share a common glycosylation site, theyare both able to traffic to the cellmembrane. None ofN8,N9, orN10 was shown to be essential because single site mutants ofeach were expressed on the cell surface and retained affinity forAc-LDL so it seems unlikely that the CD36 polypeptide foldsaround a specific glycan. The requirement for glycosylation ofCD36 for trafficking, but the redundancy observed with regardto the number and position of these sites, is more consistentwith the involvement of lectin-like molecular chaperones. Lec-tin-like chaperones have been shown to recognize the glycansof, for example, the human organic anion transporter hOAT4(45) and influenza hemagglutinin (46) and to facilitate proteinfolding in the ER before trafficking to theGolgi. This would alsoexplain why the receptor cannot be functionally expressed in

Role of Glycosylation in CD36

JUNE 12, 2009 • VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16287

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

bacteria.3 That no individual glycosylation event is essential fortrafficking of CD36 contrasts with the situation in the relatedclass B scavenger receptor SRBI in which glycosylation of twoindividual sites (neither of which are conserved in CD36) wereshown to be necessary (33).In summary, we report that 9 of the 10 putative glycosylation

sites of CD36 can be modified. We show that glycosylation ofCD36 is necessary for trafficking of the protein to the cell sur-face but not for ligand recognition. There is redundancy in boththe chemical nature and the pattern of glycosylation, suggestingthat it does not directly induce the folding of the protein but,rather, may ensure contact with the appropriate chaperoneswithin the secretory pathway. Furthermore, mutant CD36 withfewer glycosylation sites can fold, traffic to the cell surface, andbind ligand. These less complex forms of the proteinmay provemore suitable for structural studies.

Acknowledgments—We are grateful to Geeta Patel, Dinah Rahman,and Justin Lock of the MRC proteomics facility for Q-ToF MS and toCleidiane Zampronio, Antony Jones, and Helen Cooper of the Func-tional Genomics Laboratory, BirminghamUniversity, for FT-ICRMS(the Functional Genomics Laboratory is funded by Biotechnology andBiological Sciences Research Council, Swindon, United KingdomGrant 6/JIF13209).

REFERENCES1. Tandon, N. N., Kralisz, U., and Jamieson, G. A. (1989) J. Biol. Chem. 264,

7576–75832. Greenwalt, D. E.,Watt, K.W., So,O. Y., and Jiwani, N. (1990)Biochemistry

29, 7054–70593. Endemann, G., Stanton, L. W., Madden, K. S., Bryant, C. M., White, R. T.,

and Protter, A. A. (1993) J. Biol. Chem. 268, 11811–118164. Greenwalt, D. E., Lipsky, R. H., Ockenhouse, C. F., Ikeda, H., Tandon,

N. N., and Jamieson, G. A. (1992) Blood 80, 1105–11155. Harmon, C. M., and Abumrad, N. A. (1993) J. Membr. Biol. 133, 43–496. Rutella, S., Rumi, C., Di Mario, A., and Leone, G. (1997) Eur. J. Histochem.

41, Suppl. 2, 53–547. Ma, 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., and Doria, A. (2004)Hum. Mol. Genet. 13, 2197–2205

8. Febbraio, M., Podrez, E. A., Smith, J. D., Hajjar, D. P., Hazen, S. L., Hoff,H. F., Sharma, K., and Silverstein, R. L. (2000) J. Clin. Invest. 105,1049–1056

9. Serghides, L., Smith, T. G., Patel, S. N., and Kain, K. C. (2003) TrendsParasitol. 19, 461–469

10. Aitman, 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., Shoul-ders, C. C., Graf, D., St Lezin, E., Kurtz, T. W., Kren, V., Pravenec, M.,Ibrahimi, A., Abumrad, N. A., Stanton, L. W., and Scott, J. (1999) Nat.Genet. 21, 76–83

11. Love-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., andAbumrad, N. A. (2008) Hum. Mol. Genet. 17, 1695–1704

12. Yamashita, S., Hirano, K., Kuwasako, T., Janabi, M., Toyama, Y., Ishigami,M., and Sakai, N. (2007)Mol. Cell Biochem. 299, 19–22

13. Abumrad, N. A., el-Maghrabi, M. R., Amri, E. Z., Lopez, E., and Grimaldi,P. A. (1993) J. Biol. Chem. 268, 17665–17668

14. Koonen, D. P., Febbraio, M., Bonnet, S., Nagendran, J., Young, M. E.,

Michelakis, E. D., and Dyck, J. R. (2007) Circulation 116, 2139–214715. Hwang, E. H., Taki, J., Yasue, S., Fujimoto, M., Taniguchi, M., Matsunari,

I., Nakajima, K., Shiobara, S., Ikeda, T., and Tonami, N. (1998) J. Nucl.Med. 39, 1681–1684

16. Vega, M. A., Seguí-Real, B., García, J. A., Cales, C., Rodríguez, F.,Vanderkerckhove, J., and Sandoval, I. V. (1991) J. Biol. Chem. 266,16818–16824

17. Calvo, D., and Vega, M. A. (1993) J. Biol. Chem. 268, 18929–1893518. Acton, S. L., Scherer, P. E., Lodish, H. F., and Krieger, M. (1994) J. Biol.

Chem. 269, 21003–2100919. Franc, N. C., Dimarcq, J. L., Lagueux, M., Hoffmann, J., and Ezekowitz,

R. A. (1996) Immunity 4, 431–44320. Hart, K., and Wilcox, M. (1993) J. Mol. Biol. 234, 249–25321. Oquendo, P., Hundt, E., Lawler, J., and Seed, B. (1989) Cell 58, 95–10122. Gruarin, P., Thorne, R. F., Dorahy, D. J., Burns, G. F., Sitia, R., and Alessio,

M. (2000) Biochem. Biophys. Res. Commun. 275, 446–45423. Pearce, S. F., Wu, J., and Silverstein, R. L. (1994) Blood 84, 384–38924. Asch, A. S., Liu, I., Briccetti, F. M., Barnwell, J. W., Kwakye-Berko, F.,

Dokun, A., Goldberger, J., and Pernambuco, M. (1993) Science 262,1436–1440

25. Rasmussen, J. T., Berglund, L., Rasmussen,M. S., and Petersen, T. E. (1998)Eur. J. Biochem. 257, 488–494

26. Tao, N., Wagner, S. J., and Lublin, D. M. (1996) J. Biol. Chem. 271,22315–22320

27. Alessio, M., Ghigo, D., Garbarino, G., Geuna, M., and Malavasi, F. (1991)Cell. Immunol. 137, 487–500

28. Freeze, H. H. (2006) Nat. Rev. Genet. 7, 537–55129. O’Connor, S. E., and Imperiali, B. (1996) Chem. Biol. 3, 803–81230. Helenius, A. (1994)Mol. Biol. Cell 5, 253–26531. Mitra, N., Sinha, S., Ramya, T. N., and Surolia, A. (2006) Trends Biochem.

Sci. 31, 156–16332. Trombetta, E. S., and Helenius, A. (1998) Curr. Opin. Struct. Biol. 8,

587–59233. Nufer, O., Guldbrandsen, S., Degen, M., Kappeler, F., Paccaud, J. P., Tani,

K., and Hauri, H. P. (2002) J. Cell Sci. 115, 619–62834. Berglund, L., Petersen, T. E., and Rasmussen, J. T. (1996)Biochim. Biophys.

Acta 1309, 63–6835. Vinals, M., Xu, S., Vasile, E., and Krieger, M. (2003) J. Biol. Chem. 278,

5325–533236. Martin, C. A., Longman, E., Wooding, C., Hoosdally, S. J., Ali, S., Aitman,

T. J., Gutmann, D. A., Freemont, P. S., Byrne, B., and Linton, K. J. (2007)Protein Sci. 16, 2531–2541

37. Dixon, 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., and Williamson, C.(2000) Hum. Mol. Genet. 9, 1209–1217

38. Gavin, A. C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A.,Schultz, J., Rick, J. M., Michon, A. M., Cruciat, C. M., Remor, M., Hofert,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., and Superti-Furga, G. (2002) Nature 415,141–147

39. Peterman, S. M., Dufresne, C. P., and Horning, S. (2005) J. Biomol. Tech.16, 112–124

40. Klinkenberg, L. G., Webb, T., and Zitomer, R. S. (2006) Eukaryot. Cell 5,1007–1017

41. Hsieh, P., and Robbins, P. W. (1984) J. Biol. Chem. 259, 2375–238242. Jarvis, D. L. (2003) Virology 310, 1–743. Schinkel, A. H., Kemp, S., Dolle,M., Rudenko, G., andWagenaar, E. (1993)

J. Biol. Chem. 268, 7474–748144. Kulkarni, K. A., Srivastava, A., Mitra, N., Sharon, N., Surolia, A., Vijayan,

M., and Suguna, K. (2004) Proteins 56, 821–82745. Zhou, F., Xu,W., Hong, M., Pan, Z., Sinko, P. J., Ma, J., and You, G. (2005)

Mol. Pharmacol. 67, 868–87646. Hebert, D. N., Zhang, J. X., Chen,W., Foellmer, B., andHelenius, A. (1997)

J. Cell Biol. 139, 613–6233 S. J. Hoosdally, E. J. Andress, C. Wooding, C. A. Martin, and K. J. Linton, unpub-

lished data.

Role of Glycosylation in CD36

16288 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 • JUNE 12, 2009

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from

Kenneth J. LintonSarah J. Hoosdally, Edward J. Andress, Carol Wooding, Catherine A. Martin and

ROLE IN TRAFFICKING AND FUNCTIONThe Human Scavenger Receptor CD36: GLYCOSYLATION STATUS AND ITS

doi: 10.1074/jbc.M109.007849 originally published online April 15, 20092009, 284:16277-16288.J. Biol. Chem. 

  10.1074/jbc.M109.007849Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/284/24/16277.full.html#ref-list-1

This article cites 46 references, 20 of which can be accessed free at

by guest on Novem

ber 23, 2020http://w

ww

.jbc.org/D

ownloaded from