Children's Foundation Research Institute at Le Bonheur Children's Hospital, Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38103, United States.
Cell Mol Biol (Noisy-le-grand). 2022 Jan 2;67(4):163-169. doi: 10.14715/cmb/2021.67.4.18.
Siglecs, membrane-bound lectins of the sialic acid-binding immunoglobulin superfamily, inhibit immune responses by recruiting tyrosine phosphatases (e.g., SHP-1 and SHP-2) through their cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM) domain. The role of Siglecs in infection has been extensively studied, but downstream signaling through the ITIM domain remains unclear. Here, we used a GST pull-down assay to identify additional proteins associated with the ITIM domain during bacterial infection. Gdi2 bound to ITIM under normal homeostasis, but Rab1a was recruited to ITIM during bacterial infection. Western blot analysis confirmed the presence of SHP-1 and SHP-2 in eluted ITIM-associated proteins under normal homeostasis. We confirmed the association of ITIM with Gdi2 or Rab1a by transfection of corresponding expression vectors in 293T cells followed by immunoprecipitation-western blot assay. Thus, ITIM's role in the inhibition of the immune response during bacterial infection may be regulated by interaction with Gdi2 and Rab1a in addition to SHP-1 and SHP-2.
Siglecs 是唾液酸结合免疫球蛋白超家族中的膜结合凝集素，通过其细胞质免疫受体酪氨酸抑制基序 (ITIM) 结构域招募酪氨酸磷酸酶（例如 SHP-1 和 SHP-2）来抑制免疫反应。Siglecs 在感染中的作用已被广泛研究，但 ITIM 结构域的下游信号仍然不清楚。在这里，我们使用 GST 下拉测定法来鉴定在细菌感染期间与 ITIM 结构域相关的其他蛋白。Gdi2 在正常的体内平衡下与 ITIM 结合，但 Rab1a 在细菌感染期间被招募到 ITIM 上。Western blot 分析证实，在正常的体内平衡下，SHP-1 和 SHP-2 存在于洗脱的 ITIM 相关蛋白中。我们通过在 293T 细胞中转染相应的表达载体，然后进行免疫沉淀-Western blot 分析，证实了 ITIM 与 Gdi2 或 Rab1a 的关联。因此，ITIM 在细菌感染期间抑制免疫反应的作用可能通过与 Gdi2 和 Rab1a 的相互作用来调节，除了 SHP-1 和 SHP-2 之外。
