Sulfo-N-succinimidyl Oleate (SSO) Inhibits Fatty Acid Uptake and Signaling for Intracellular Calcium via Binding CD36 Lysine 164

FAT/CD36 is a multifunctional glycoprotein that facilitates long-chain fatty acid (FA) uptake by cardiomyocytes and adipocytes and uptake of oxidized low density lipoproteins (oxLDL) by macrophages. CD36 also mediates FA-induced signaling to increase intracellular calcium in various cell types. The...

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Bibliographic Details
Published in:The Journal of biological chemistry 2013-05, Vol.288 (22), p.15547-15555
Main Authors: Kuda, Ondrej, Pietka, Terri A., Demianova, Zuzana, Kudova, Eva, Cvacka, Josef, Kopecky, Jan, Abumrad, Nada A.
Format: Article
Language:English
Subjects:
CD36
Fatty Acid
Fatty Acid Uptake
Glycerophospholipid
Lipid Transport
Lipids
Metabolic Regulation
SSO
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Summary:FAT/CD36 is a multifunctional glycoprotein that facilitates long-chain fatty acid (FA) uptake by cardiomyocytes and adipocytes and uptake of oxidized low density lipoproteins (oxLDL) by macrophages. CD36 also mediates FA-induced signaling to increase intracellular calcium in various cell types. The membrane-impermeable sulfo-N-hydroxysuccinimidyl (NHS) ester of oleate (SSO) irreversibly binds CD36 and has been widely used to inhibit CD36-dependent FA uptake and signaling to calcium. The inhibition mechanism and whether SSO modification of CD36 involves the FA-binding site remain unexplored. CHO cells expressing human CD36 were SSO-treated, and the protein was pulled down, deglycosylated, and resolved by electrophoresis. The CD36 band was extracted from the gel and digested for analysis by mass spectrometry. NHS derivatives react with primary or secondary amines on proteins to yield stable amide or imide bonds. Two oleoylated peptides, found only in SSO-treated samples, were identified with high contribution and confidence scores as carrying oleate modification of Lys-164. Lysine 164 lies within a predicted CD36 binding domain for FA and oxLDL. CHO cells expressing CD36 with mutated Lys-164 had impaired CD36 function in FA uptake and FA-induced calcium release from the endoplasmic reticulum, supporting the importance of Lys-164 for both FA effects. Furthermore, consistent with the importance of Lys-164 for oxLDL binding, SSO inhibited oxLDL uptake by macrophages. In conclusion, SSO accesses Lys-164 in the FA-binding site on CD36, and initial modeling of this site is presented. The data suggest competition between FA and oxLDL for access to the CD36 binding pocket. Background: Mechanism of CD36 inhibition by sulfo-N-succinimidyl oleate (SSO) was explored using mass spectrometry and mutagenesis. Results: SSO binds lysine 164 and inhibits uptake of fatty acids and oxLDL. Conclusion: Lysine 164 is important for CD36-mediated fatty acid uptake and Ca2+ signaling. Significance: Fatty acids and oxLDL bind to the same site within a hydrophobic pocket of CD36 shared by several lipid ligands.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M113.473298