Alborzian Deh Sheikh Amin, Akatsu Chizuru, Imamura Akihiro, Abdu-Allah Hajjaj H M, Takematsu Hiromu, Ando Hiromune, Ishida Hideharu, Tsubata Takeshi
Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.
Department of Applied Bio-organic Chemistry, Gifu University, Gifu 501-1193, Japan.
Biochem Biophys Res Commun. 2018 Jan 1;495(1):854-859. doi: 10.1016/j.bbrc.2017.11.086. Epub 2017 Nov 14.
Lectins expressed on the cell surface are often bound and regulated by the membrane molecules containing the glycan ligands on the same cell (cis-ligands). However, molecular nature and function of cis-ligands are generally poorly understood partly because of weak interaction between lectins and glycan ligands. Cis-ligands are most extensively studied in CD22 (also known as Siglec-2), an inhibitory B lymphocyte receptor specifically recognizing α2,6 sialic acids. CD22, CD45 and IgM are suggested to be ligands of CD22. Here we labeled molecules in the proximity of CD22 in situ on B cell surface using biotin-tyramide. Molecules including CD22, CD45 and IgM were labeled in wild-type but not ST6GalI B cells that lack α2,6 sialic acids, indicating that these molecules associate with CD22 by lectin-glycan interaction, and are therefore cis-ligands. In ST6GalI B cells, these cis-ligands are located in a slightly more distance from CD22. Thus, the lectin-glycan interaction recruits cis-ligands already located in the relative proximity of CD22 through non-lectin-glycan interaction to the close proximity. Moreover, cis-ligands are labeled in Cmah B cells that lack Neu5Gc preferred by mouse CD22 as efficiently as in wild-type B cells, indicating that very low affinity lectin-glycan interaction is sufficient for recruiting cis-ligands, and can be detected by proximity labeling. Thus, proximity labeling with tyramide appears to be a useful method to identify cis-ligands and to analyze their interaction with the lectins.
细胞表面表达的凝集素通常会被同一细胞上含有聚糖配体的膜分子（顺式配体）结合并调节。然而，由于凝集素与聚糖配体之间的相互作用较弱，顺式配体的分子性质和功能通常了解甚少。顺式配体在CD22（也称为Siglec-2）中研究最为广泛，CD22是一种特异性识别α2,6唾液酸的抑制性B淋巴细胞受体。CD22、CD45和IgM被认为是CD22的配体。在这里，我们使用生物素酪胺原位标记B细胞表面CD22附近的分子。包括CD22、CD45和IgM在内的分子在野生型B细胞中被标记，但在缺乏α2,6唾液酸的ST6GalI B细胞中未被标记，这表明这些分子通过凝集素-聚糖相互作用与CD22结合，因此是顺式配体。在ST6GalI B细胞中，这些顺式配体与CD22的距离稍远。因此，凝集素-聚糖相互作用通过非凝集素-聚糖相互作用将已经位于CD22相对附近的顺式配体招募到紧密附近。此外，在缺乏小鼠CD22偏好的Neu5Gc的Cmah B细胞中，顺式配体的标记效率与野生型B细胞一样高，这表明极低亲和力的凝集素-聚糖相互作用足以招募顺式配体，并且可以通过邻近标记检测到。因此，酪胺邻近标记似乎是一种识别顺式配体并分析它们与凝集素相互作用的有用方法。
