Recombinant glutathione S-transferase/CD36 fusion proteins define an oxidized low density lipoprotein-binding domain.
CD36 is a multifunctional cell-surface receptor that binds adhesion molecules such as thrombospondin-1 and collagen and modified lipids and/or lipoproteins. It participates in cellular uptake of photoreceptor outer segments and scavenging of apoptotic cells and oxidized low density lipoprotein (Ox-LDL). Recognition and internalization of Ox-LDL by mononuclear phagocytes may play an important role in the development of atherosclerotic lesions. We have utilized a series of recombinant bacterial glutathione S-transferase/CD36 fusion proteins that span nearly all of the CD36 molecule to characterize the structural domain on CD36 that recognizes Ox-LDL. We found that the Ox-LDL-binding domain is different from the thrombospondin-1-binding domain located at amino acids 93-120. A fusion protein containing the region extending from amino acids 5 to 143 formed specific, saturable, and reversible complexes with Ox-LDL. As with intact CD36, binding was blocked by excess unlabeled Ox-LDL and antibodies to CD36. The stoichiometry and affinity of the fusion protein for Ox-LDL were similar to those of the intact protein. We also demonstrated that this fusion protein competitively inhibited binding of Ox-LDL to purified platelet CD36 and to CD36 expressed on peripheral blood monocytes and CD36 cDNA-transfected melanoma cells. The use of smaller peptides and fusion proteins including those spanning amino acids 28-93 and 5-93 has further narrowed the binding site to a region from amino acids 28 to 93, although participation of a sequence in the noncontiguous region 120-155 cannot be excluded. This study, for the first time, demonstrates unique regions of the scavenger receptor CD36 that bind the Ox-LDL ligand. Our structural analysis of the receptor provides information as to potential control of the trafficking of modified lipoproteins into the blood vessel wall.
Pearce
S F
SF
Department of Medicine, Division of Hematology-Oncology, Cornell University Medical College, New York, New York 10021, USA.
Roy
P
P
Nicholson
A C
AC
Hajjar
D P
DP
Febbraio
M
M
Silverstein
R L
RL
D001665:Binding Sites; D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism; D018955:CD36 Antigens / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019700:Thrombospondin 1 / Q000378:metabolism
D018955:CD36 Antigens; D008077:Lipoproteins, LDL; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019700:Thrombospondin 1; C099252:oxidized low density lipoprotein; D005982:Glutathione Transferase