Proximity labeling of cis-ligands of CD22/Siglec-2 reveals stepwise α2,6 sialic acid-dependent and -independent interactions.
Proximity labeling of cis-ligands of CD22/Siglec-2 reveals stepwise α2,6 sialic acid-dependent and -independent interactions.
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CD22/Siglec-2 顺式配体的邻近标记揭示了逐步的 α2,6 唾液酸依赖性和非依赖性相互作用。
DOI：
10.1016/j.bbrc.2017.11.086
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Tsubata T
中科院分区：
文献类型：
--
作者：
Alborzian Deh Sheikh A;Akatsu C;Imamura A;Abdu-Allah HHM;Takematsu H;Ando H;Ishida H;Tsubata T
在细胞表面表达的凝集素通常被同一细胞上含有聚糖配体（顺式配体）的膜分子结合和调节。然而，顺式配体的分子性质和功能通常知之甚少，部分原因是凝集素和聚糖配体之间的弱相互作用。顺式配体在CD 22（也称为Siglec-2）中的研究最为广泛，后者是一种特异性识别α 2，6唾液酸的抑制性B淋巴细胞受体。CD 22、CD 45和IgM可能是CD 22的配体。在这里，我们使用生物素-酪胺在B细胞表面原位标记CD 22附近的分子。包括CD 22、CD 45和IgM在内的分子在野生型中被标记，但在缺乏α 2，6唾液酸的ST 6 GalI −/-B细胞中未被标记，表明这些分子通过凝集素-聚糖相互作用与CD 22结合，因此是顺式配体。在ST 6 GalI −/-B细胞中，这些顺式配体位于距离CD 22稍远的位置。因此，凝集素-聚糖相互作用通过非凝集素-聚糖相互作用募集已经位于CD 22相对邻近的顺式配体。此外，顺式配体在缺乏小鼠CD 22偏好的Neu 5Gc的Cmah−/-B细胞中与在野生型B细胞中一样有效地被标记，表明非常低亲和力的凝集素-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。因此，接近标记与酪胺似乎是一个有用的方法来确定顺式配体和分析它们与凝集素的相互作用。
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.