Sulfo-N-succinimidyl Oleate (SSO) Inhibits Fatty Acid Uptake and Signaling for Intracellular Calcium via Binding CD36 Lysine 164
Sulfo-N-succinimidyl Oleate (SSO) Inhibits Fatty Acid Uptake and Signaling for Intracellular Calcium via Binding CD36 Lysine 164
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DOI：
10.1074/jbc.m113.473298
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发表时间：
2013-05-31
影响因子：
4.8
通讯作者：
Abumrad, Nada A.
中科院分区：
文献类型：
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作者：
Kuda, Ondrej;Pietka, Terri A.;Abumrad, Nada A.
FAT/CD36是一种多功能糖蛋白，促进心肌细胞和脂肪细胞对长链脂肪酸（FA）的摄取，以及巨噬细胞对氧化低密度脂蛋白（oxLDL）的摄取。CD36还介导fa诱导的信号传导，在各种细胞类型中增加细胞内钙。膜不渗透性油酸磺酸- n -羟基琥珀酰亚胺基（NHS）酯（SSO）不可逆地结合CD36，并被广泛用于抑制CD36依赖性FA摄取和钙信号传导。CD36的抑制机制以及单点登录修饰是否涉及fa结合位点尚不清楚。表达人CD36的CHO细胞被sso处理，蛋白被拉下，去糖基化，并通过电泳分离。从凝胶中提取CD36条带并进行质谱分析。NHS衍生物与蛋白质上的伯胺或仲胺反应，产生稳定的酰胺或亚胺键。仅在sso处理的样品中发现的两种油酸化肽被鉴定为具有高贡献和置信度分数的携带Lys-164的油酸修饰。赖氨酸164位于FA和oxLDL预测的CD36结合域内。表达CD36和突变Lys-164的CHO细胞在FA摄取和FA诱导的钙从内质网释放中的CD36功能受损，支持Lys-164对两种FA效应的重要性。此外，与Lys-164对oxLDL结合的重要性一致，SSO抑制了巨噬细胞对oxLDL的摄取。综上所述，SSO可以访问CD36上fa结合位点的Lys-164，并对该位点进行了初步建模。这些数据表明FA和oxLDL在进入CD36结合口袋方面存在竞争。
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.