Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jun 12;284(24):16277-16288.
doi: 10.1074/jbc.M109.007849. Epub 2009 Apr 15.

The Human Scavenger Receptor CD36: glycosylation status and its role in trafficking and function

Sarah J Hoosdally  1 , Edward J Andress  1 , Carol Wooding  1 , Catherine A Martin  1 , Kenneth J Linton  2
Affiliations

The Human Scavenger Receptor CD36: glycosylation status and its role in trafficking and function

Sarah J Hoosdally et al. J Biol Chem. .

Abstract

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.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Purification of CD36–12His by affinity chromatography and deglycosylation by PNGase F. A, SDS-polyacrylamide gel stained with colloidal blue showing protein fractions during the purification of wild type CD36–12His from baculoviral-infected Sf21 cells. Lane 1, 50 μg of crude membrane fraction (0.1% of starting material); lane 2, OG-solubilized membrane proteins (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 to remove proteins non-specifically bound (2% by volume); lanes 9–12, elution fractions in 250 mm imidazole (2% by volume). B, the electrophoretic mobility of purified recombinant wild type CD36 (lane 1) was compared with CD36 after deglycosylation by PNGase F (lane 2) and non-glycosylatable CD36non-g purified from protein aggregates (lane 3).
FIGURE 2.
FIGURE 2.
The affinity of CD36 for ligand is not affected directly by the nature or pattern of glycosylation. Interaction of CD36 proteins with Ac-LDL was measured using increasing concentrations of BODIPY Ac-LDL to core-glycosylated, wild type (wt) CD36 purified from Sf21 insect cells and immobilized on a solid phase (A), mature glycosylated, wild type CD36 on the surface 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 to Equation 1; the Kd quoted represents the mean ± S.E. (μg/ml) from at least three independent experiments.
FIGURE 3.
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 that N7 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 MS spectrum of similarly treated wild type CD36 showing that N6 can be glycosylated. The sequence of the tryptic digest fragments with the putative glycosylated asparagine underlined and the complete y and b-series ions generated by the ionization of the PNGase-F-deglycosylated and -deamidated fragment are shown. 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 MS spectrum, 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 monoisotopic parent 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 deviations from the expected ion mass, see supplemental Fig. 1.
FIGURE 4.
FIGURE 4.
Electrophoretic mobility shift analysis to test glycosylation status of CD36. A, whole cell lysates (50 μg of protein) from transiently transfected HEK293T 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, CD36N8–10+N7; lane 3, CD36N8–10; lane 4, CD36N8–10+N3; lane 5, CD36N8–10+N4; lane 6, CD36N8–10+N6; lane 7, CD36N8–10; lane 8, CD36N8–10+N7; lane 9, CD36N8–10+N2. B and C, analysis of CD36N8–10 probed with mAb1955 (B) and P-glycoprotein probed with mAbC219 (C) after tunicamycin treatment of the transfected cells to inhibit glycosylation: lane 1, untreated sample; lane 2, treated with PNGase F; lane 3, treated with tunicamycin; lane 4, treated with tunicamycin and PNGase F.
FIGURE 5.
FIGURE 5.
Non-glycosylatable CD36non-g fails to traffic to the plasma membrane. A, immunoblot analysis probing with mAb1955 confirms CD36non-g is translated when expressed transiently in HEK293T cells; the lower molecular weight species in the non-g lane is not always observed and is likely to be a degradation product. B, whole cell flow cytometry analysis using an antibody (mAb1258) that recognizes an epitope in the extracellular loop of CD36 indicates that CD36non-g fails to traffic to the plasma membrane. Wild type CD36 is shown in black, CD36non-g shown in blue, and untransfected cells shown in red. PE, R-Phycoerythrin.
FIGURE 6.
FIGURE 6.
Cell surface expression of CD36 mutants. Wild type and mutated CD36 and P-glycoprotein were expressed transiently in HEK293T cells, and flow cytometry was used to measure the cell surface expression using saturating amounts of antibody. Wild type CD36, which traffics efficiently, was used as the positive control (black trace, where shown), and CD36non-g, which fails to traffic, was used as the negative control (blue trace, where shown) for all CD36 experiments. PE, R-Phycoerythrin. A, cell surface expression of CD36N7–10 (green) and CD36N1–7 (red). B, CD36N7–10 (green) and CD36N8–10 (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-glycoprotein (Pgp) in untreated (red) and tunicamycin-treated cells (green). F, cell surface expression of CD36N1–8,10 (red), CD36N1–9 (green), and CD36N1–7,9,10 (light blue). G, cell surface expression of CD36N2,8–10.
FIGURE 7.
FIGURE 7.
Some trafficking-competent CD36 isoforms are only core-glycosylated. 50 μg of whole cell protein lysates from transiently transfected HEK293T cells treated with either 1 unit of Endo H or PNGase F were analyzed by immunoblotting probing with mAb1955. A, wild type CD36: lane 1, untreated; lane 2, treated with Endo 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 with PNGase 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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Tandon N. N., Kralisz U., Jamieson G. A. ( 1989) J. Biol. Chem. 264, 7576– 7583 - PubMed
    1. Greenwalt D. E., Watt K. W., So O. Y., Jiwani N. ( 1990) Biochemistry 29, 7054– 7059 - PubMed
    1. Endemann G., Stanton L. W., Madden K. S., Bryant C. M., White R. T., Protter A. A. ( 1993) J. Biol. Chem. 268, 11811– 11816 - PubMed
    1. Greenwalt D. E., Lipsky R. H., Ockenhouse C. F., Ikeda H., Tandon N. N., Jamieson G. A. ( 1992) Blood 80, 1105– 1115 - PubMed
    1. Harmon C. M., Abumrad N. A. ( 1993) J. Membr. Biol. 133, 43– 49 - PubMed

Publication types

MeSH terms

LinkOut - more resources