title: Sulfo-N-succinimidyl oleate (SSO) inhibits fatty acid uptake and signaling for intracellular calcium via binding CD36 lysine 164: SSO also inhibits oxidized low density lipoprotein uptake by macrophages.
abstract: 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.
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 14220 Praha 4, Czech Republic. kuda@biomed.cas.cz
authors.forename: Ondrej
authors.initials: O
authors.lastname: Kuda
authors.forename: Terri A
authors.initials: TA
authors.lastname: Pietka
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Demianova
authors.forename: Eva
authors.initials: E
authors.lastname: Kudova
authors.forename: Josef
authors.initials: J
authors.lastname: Cvacka
authors.forename: Jan
authors.initials: J
authors.lastname: Kopecky
authors.forename: Nada A
authors.initials: NA
authors.lastname: Abumrad
mesh_terms: D000818:Animals; D001665:Binding Sites; D018955:CD36 Antigens / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000235:genetics / Q000378:metabolism*; D009829:Oleic Acids / Q000378:metabolism* / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D013388:Succinimides / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D018955:CD36 Antigens; D008077:Lipoproteins, LDL; D009829:Oleic Acids; D013388:Succinimides; C099252:oxidized low density lipoprotein; C120556:sulfo-N-succinimidyl oleate
keywords: CD36; Fatty Acid; Fatty Acid Uptake; Glycerophospholipid; Lipid Transport; Lipids; Metabolic Regulation; SSO