Institute of Biomedical Sciences, Academia Sinica, No.128, Sec.II, Academy Road, Taipei, 115, Taiwan.
Institute of Biological Chemistry, Academia Sinica, Taipei, 115, Taiwan.
J Biomed Sci. 2021 Jan 5;28(1):5. doi: 10.1186/s12929-020-00698-z.
The accumulation of lipid-laden macrophages, foam cells, within sub-endothelial intima is a key feature of early atherosclerosis. Siglec-E, a mouse orthologue of human Siglec-9, is a sialic acid binding lectin predominantly expressed on the surface of myeloid cells to transduce inhibitory signal via recruitment of SH2-domain containing protein tyrosine phosphatase SHP-1/2 upon binding to its sialoglycan ligands. Whether Siglec-E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established.
ApoE-deficient (apoE) and apoE/Siglec-E-double deficient (apoE/Siglec-E) mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were assessed. Modified low-density lipoprotein (LDL) uptake and foam cell formation in wild type (WT) and Siglec-E- peritoneal macrophages were examined in vitro. Potential Siglec-E-interacting proteins were identified by proximity labeling in conjunction with proteomic analysis and confirmed by coimmunoprecipitation experiment. Impacts of Siglec-E expression and cell surface sialic acid status on oxidized LDL uptake and signaling involved were examined by biochemical assays.
Here we show that genetic deletion of Siglec-E accelerated atherosclerosis without affecting lipid profile in apoE mice. Siglec-E deficiency promotes foam cell formation by enhancing acetylated and oxidized LDL uptake without affecting cholesterol efflux in macrophages in vitro. By performing proximity labeling and proteomic analysis, we identified scavenger receptor CD36 as a cell surface protein interacting with Siglec-E. Further experiments performed in HEK293T cells transiently overexpressing Siglec-E and CD36 and peritoneal macrophages demonstrated that depletion of cell surface sialic acids by treatment with sialyltransferase inhibitor or sialidase did not affect interaction between Siglec-E and CD36 but retarded Siglec-E-mediated inhibition on oxidized LDL uptake. Subsequent experiments revealed that oxidized LDL induced transient Siglec-E tyrosine phosphorylation and recruitment of SHP-1 phosphatase in macrophages. VAV, a downstream effector implicated in CD36-mediated oxidized LDL uptake, was shown to interact with SHP-1 following oxidized LDL treatment. Moreover, oxidized LDL-induced VAV phosphorylation was substantially lower in WT macrophages comparing to Siglec-E counterparts.
These data support the protective role of Siglec-E in atherosclerosis. Mechanistically, Siglec-E interacts with CD36 to suppress downstream VAV signaling involved in modified LDL uptake.
富含脂质的巨噬细胞泡沫细胞在内皮下内膜中的积累是早期动脉粥样硬化的一个关键特征。Siglec-E 是人类 Siglec-9 的小鼠同源物，是一种主要表达在髓样细胞表面的唾液酸结合凝集素，通过与其唾液酸化糖链配体结合后募集含 SH2 结构域的蛋白酪氨酸磷酸酶 SHP-1/2 来传递抑制信号。Siglec-E 在巨噬细胞上的表达是否影响泡沫细胞的形成和动脉粥样硬化仍有待确定。
载脂蛋白 E 缺陷（apoE）和 apoE/Siglec-E 双缺陷（apoE/Siglec-E）小鼠给予高脂肪饮食 3 个月，评估其血脂谱和动脉粥样硬化严重程度。体外检测野生型（WT）和 Siglec-E-腹腔巨噬细胞对修饰的低密度脂蛋白（LDL）的摄取和泡沫细胞形成。通过邻近标记结合蛋白质组学分析鉴定潜在的 Siglec-E 相互作用蛋白，并通过共免疫沉淀实验进行验证。通过生化测定研究 Siglec-E 表达和细胞表面唾液酸状态对氧化型 LDL 摄取和相关信号转导的影响。
本研究表明，Siglec-E 的基因缺失加速了 apoE 小鼠的动脉粥样硬化，而不影响血脂谱。Siglec-E 缺失通过增强乙酰化和氧化型 LDL 的摄取促进泡沫细胞形成，而不影响巨噬细胞中的胆固醇流出。通过邻近标记和蛋白质组学分析，我们鉴定出清道夫受体 CD36 是与 Siglec-E 相互作用的细胞表面蛋白。在 HEK293T 细胞瞬时过表达 Siglec-E 和 CD36 以及腹腔巨噬细胞中进行的进一步实验表明，用唾液酸转移酶抑制剂或神经氨酸酶处理以耗尽细胞表面唾液酸并不影响 Siglec-E 与 CD36 之间的相互作用，但会阻碍 Siglec-E 介导的氧化型 LDL 摄取抑制。随后的实验表明，氧化型 LDL 诱导巨噬细胞中 Siglec-E 的酪氨酸磷酸化和 SHP-1 磷酸酶募集。VAV，一种涉及 CD36 介导的氧化型 LDL 摄取的下游效应物，在氧化型 LDL 处理后被证明与 SHP-1 相互作用。此外，与 Siglec-E 对应物相比，WT 巨噬细胞中氧化型 LDL 诱导的 VAV 磷酸化明显降低。
这些数据支持 Siglec-E 在动脉粥样硬化中的保护作用。在机制上，Siglec-E 与 CD36 相互作用，抑制涉及修饰的 LDL 摄取的下游 VAV 信号转导。
