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 to be linked by in bovine L. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar), and the cysteines, two at are N. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), to the ditopic topological CD36 is also modified by N-linked glycosylation, which for the that the protein with an mass of on (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, M. D. G. M. F. Cell. 1991; PubMed Scopus Google Scholar) a mass for the of glycosylation is a of extracellular and and in the glycosylation to a wide range of diseases known as disorders of glycosylation Nat. Genet. PubMed Scopus Google Scholar). can be for folding of proteins B. Chem. Biol. 1996; Full Text PDF PubMed Scopus Google Scholar, A. Mol. Biol. Cell. 1994; PubMed Scopus Google Scholar) by the of the N. S. A. Trends Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar) an for such as A. Biol. 1998; PubMed Scopus Google Scholar). has also been to be for the trafficking of certain for S. M. F. K. J. Cell PubMed Google Scholar). The glycosylation of bovine has been determined with putative sites to be glycosylated L. Biophys. 1996; PubMed Scopus Google Scholar). Human and bovine CD36 are are and glycosylation sites has 10 putative glycosylation In the SRBI, which is to CD36, are putative N-linked glycosylation of which are with the of of the sites in SRBI in an type protein that are with two and for trafficking of of these two in cell surface expression of the protein M. S. E. Krieger M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar); however, neither site is conserved in CD36. of the role of glycosylation of CD36, we used and analysis spectrometry and to which glycosylation sites are in CD36. and ligand binding studies with these proteins also the role of glycosylation and site in the trafficking and function of the The n-octyl-β-d-glucopyranoside was The was and LDL was the fatty and were were and peptide N-glycosidase F and endoglycosidase H were The and and both CD36 for in but the and and sites were the and of the CD36 by chain and two sites were used to CD36 the a C.A. E. C. S. T.J. B. 2007; PubMed Scopus Google Scholar) to The was used to a the as For expression in mammalian cells, the modified CD36 was the sequence in C.A. E. C. S. T.J. B. 2007; PubMed Scopus Google Scholar) the and to was on the to the were to the in putative glycosylation site with a for The sequence of was by The of for the to mutants cells were in modified with bovine serum at in The cells were with type and as N. J. E. J. C. M. G. E. C.F. C. Hum. Mol. Genet. 2000; PubMed Scopus Google Scholar). the cells were with acid and for a for in and was of Spodoptera frugiperda 21 cells were in at with at at a density of were with type non-glycosylated CD36 a of of at the was to a density of with the cells were by at for 10 and in and The cells were in 10 of and at the cells were at by at The was at for 10 at to the large and cells. The was and at in a for at to The membrane was in with and at protein of the membrane were determined by were by at for in a at The of type CD36 were in and at by in a and for at The was by at for in a at was in was with in the of was to the of type CD36 and with the a of with for at The was with and a of in and to proteins to the type CD36 was The was by with was used for of non-glycosylated CD36 in the and in the and The protein was with a as For in mass of type CD36 was at for 10 and F for at as 10 of purified type CD36 and were separated by and with The protein were and with for the mass spectroscopy for the Fourier transform ion cyclotron resonance were and the peptide was by as M. R. P. M. A. J. A.M. C.M. M. C. M. M. H. A. Klein K. M. D. T. V. A. S. B. C. M.A. A. E. V. G. M. T. P. B. B. G. G. PubMed Scopus Google Scholar) Fourier transform mass spectrometry mass as S. J. Google Scholar). and were in with the data the and data cells were in and fatty and at cells of was to of cells and for at of monoclonal antibody was for The cells were at for at and in of The was and the cells were in of of antibody to was to the cells and in the for at The cells were by and as and in of cells of and were for surface expression and The cell surface expression of was by The cells a likely on the individual cell was about The of these cell to but the of CD36 consistent to the and type CD36 and The expression of the receptor with the and the was and in The expression of CD36 proteins was with the expression of type and proteins to for in on different in expression was by the surface expression of the type protein by the surface expression of the The was by the of the by the of the of the individual mutants, as T. Cell. PubMed Scopus Google Scholar). For purified of type was to in of protein binding and and at with protein was and was with of ligand binding fatty of were in to a of and at in the with for ligand was and the were with of fatty of was the a binding of was of the of to For the cell cells were of and with as the cells were with of with fatty was to and at for with of were in fatty to a of and at in the with for ligand was and the were with of of was the as and of the specific binding by of the of to cells. were and binding data were by which binding of ligand to a class of binding site B is ligand is of ligand and is the of ligand giving binding and a of the of cell with of H of F as were separated by and to were with and as to by as Using the sequence is amino acid and for were identified as sites of analysis of purified protein can be used to the of these type CD36 was with a carboxyl-terminal and expressed in cells a were in cell membrane and the CD36 was purified by Although cells the mammalian glycosylation sites P. J. Biol. Chem. Full Text PDF PubMed Google Scholar), not on the 2003; PubMed Scopus Google Scholar). It to that the CD36 in the was for The purified protein was on with and its for was determined The for of the receptor purified cells was not different the for the protein expressed on the surface of mammalian cells the purified protein its and ligand binding and the specific nature of the is not for ligand 2The of CD36 for ligand is not by the nature pattern of glycosylation. of CD36 proteins with was of to core-glycosylated, type CD36 purified cells and on a mature type CD36 on the surface of cells and on the surface of cells is and is to the the at of to CD36 type CD36 type CD36 in in in in in in a of the CD36 purified cells with F increased the mobility of the protein The mass of of the 10 putative glycosylation sites are The CD36 with mobility to a of the protein consistent with N-linked of type CD36. of a protein by F the N-linked the peptide chain and the glycosylated to an This results in a mass of for as with the mass of the protein a mass of a is to and to of the of the and increased mass to used this mass with the to the glycosylation of the protein by mass spectrometry of type CD36 of which the putative N-linked glycosylation In are to different carboxyl-terminal and and analysis of the mass and of these the amino acid sequence of the peptide For example, putative glycosylation and with at the amino of the is at the amino the has been and to an acid The with and are on the and both are for acid at of of the type by glycosylation. glycosylation site is in the of the both and can this site is The with is for acids in both and and the with and an acid in that is glycosylated in type CD36 with that are not within glycosylation sites show of mass indicating that is not on type CD36, in the of F to putative glycosylation of CD36, not to be were This is consistent with glycosylation of the relevant as the of is low to and the likely of glycosylation. mass spectrometry of the protein identified of the and was to that and were as glycosylation sites data of the and their mass the are in The putative glycosylation sites were in large mass for mass for to be by to to the glycosylation of these putative of the protein and identified that can also be glycosylated the a However, not this mass indicating that is to be of both these are in F and the and was not This is most likely to a low for the which has an with not in for by mass electrophoretic mobility of CD36 with and the site was used to the glycosylation of these putative sites and of the utilization of that the in electrophoretic mobility be in a less CD36 with glycosylation we established that with the carboxyl-terminal sites to the cell surface for of this with an mass of on of mutants were on the to the utilization of putative glycosylation sites and sites were the minimally glycosylated to and mutants, with were expressed in cells, and the electrophoretic mobility of the proteins was by and by with It was that the putative glycosylation site is in of the mutants, the electrophoretic mobility of this protein is likely to be that of a was by to be glycosylated in the type was with and found to the The that and are glycosylated as and with of the CD36 proteins with the mobility that the on and were for the electrophoretic in In of site in the in glycosylation of the protein as by the of protein in of the of which are a in the by with F. Although we the that of the of sites the and that is consistent with the data on the type is that is as a glycosylation In these we found no to is modified by glycosylation. with the mass spectrometry is that 9 of the 10 putative glycosylation sites can be modified by the mutants and of that have been to in a the role of glycosylation in the of CD36, we 10 putative glycosylation the conserved with by to The was expressed in the cell is by cells as by with the monoclonal but cells were by for surface expression data not is that to to the plasma membrane which N-linked glycosylation sites are for CD36 to the cell we glycosylation sites of mutants Flow was used to the of cell surface expression of the proteins expressed in cells. not of and were used to of surface analysis of two mutants with an pattern of glycosylation sites that both the plasma membrane but were expressed to different and of type CD36 the of of the of antibody binding to cells is the binding with This indicated two was likely that was both an and a carboxyl-terminal of glycosylation sites that supported trafficking to be the key the low of expression of both mutants that sites in both the and carboxyl-terminal were likely to be necessary to expression of the cell surface expression of CD36 to by expression are the are the are the in a which of the glycosylation sites of CD36 of which are modified by N-linked were necessary for the protein to the cell sites were of The expression of was the as but to to the cell a in trafficking with and to the plasma membrane of the mutants were by the monoclonal antibody which both the type and the non-glycosylated of the protein to that the glycosylation site mutants were by the cells of surface not data the importance of and in the however, a with sites and present to to the cell indicating that these sites can an in the of a heavily glycosylated that not appear to be modified by N-linked glycosylation, is a glycosylation site density and trafficking within the mutants with of the glycosylation sites of appear on the cell mutants with sites are to This of glycosylation sites the of the sites to the trafficking however, their on the trafficking with the carboxyl-terminal that sites and were the that can be expressed at the cell surface and the that no glycosylation site with that of the glycosylation sites is essential for CD36 to to the cell and analysis of mutants and of that was not for trafficking of CD36 to the plasma the surface expression of was the as The of and to at the cell surface that sites and of which were by to be modified by N-linked were essential for expression and that the glycosylation sites in were the in the carboxyl that cell surface proteins with two of these glycosylation and of were to to the cell membrane Although the carboxyl-terminal glycosylation sites were to trafficking of an non-glycosylated CD36, the individual sites have a on the of trafficking glycosylation sites are This of the sites was to and to the cell membrane with and of the type protein of the of mutants of the carboxyl-terminal sites an type to these sites to the trafficking of the and to the cell membrane on and that of the of type CD36 of with the trafficking of which carboxyl-terminal sites are and which is present at the cell surface at that of the of the type is a that the carboxyl-terminal sites function This is the of the individual mutants with the type range and but the surface expression of the is The the of the by the of the of the individual mutants, the mutants are the of a the sites and trafficking of the protein the of glycosylation we to and their to to the plasma membrane of site and site on the trafficking of the consistent with that no on trafficking of the and that is not as a glycosylation of the sites the trafficking of with sites and the of the cell surface expression of the type protein the surface expression of is in