Novel Anti-carbohydrate Antibodies Reveal the Cooperative Function of Sulfated N- and O-Glycans in Lymphocyte Homing
Novel Anti-carbohydrate Antibodies Reveal the Cooperative Function of Sulfated N- and O-Glycans in Lymphocyte Homing
复制标题
DOI：
10.1074/jbc.m110.167296
复制
发表时间：
2010-12-24
影响因子：
4.8
通讯作者：
Kawashima, Hiroto
中科院分区：
文献类型：
--
作者：
Hirakawa, Jotaro;Tsuboi, Koichiro;Kawashima, Hiroto
细胞表面聚糖在免疫细胞运输和免疫中发挥着关键作用。在这里，我们提出了一种使用基因靶向小鼠产生抗碳水化合物单克隆抗体（mAb）的有效方法，并描述了淋巴细胞归巢中的关键聚糖。我们用磺基转移酶过表达的中国仓鼠卵巢细胞免疫磺基转移酶 GlcNAc6ST-1 和 GlcNAc6ST-2 双缺陷小鼠，并产生了两种 mAb，称为 S1 和 S2。 S1 和 S2 都与人类和野生型小鼠淋巴器官中的高内皮微静脉 (HEV) 结合，但在双缺陷小鼠的淋巴器官中则不然。聚糖阵列分析表明，S1 和 S2 均特异性结合 6-磺基唾液酸基 Lewis X 及其去岩藻基化结构。有趣的是，S2 抑制淋巴细胞归巢至外周淋巴结 95%，而 S1 仅抑制 25%。 S2 还显着抑制接触超敏反应和 L-选择素依赖性白细胞对 HEV 的粘附。免疫组织化学和蛋白质印迹分析表明，S1 优先结合 HEV 中的硫酸化 O-聚糖，而 S2 结合硫酸化 N-和 O-聚糖。此外，在缺乏硫酸化O-聚糖的突变小鼠中，S2强烈抑制N-聚糖依赖性残余淋巴细胞归巢，表明硫酸化N-和O-聚糖在淋巴细胞归巢中的重要性。因此，通过一种新方法产生的两种单克隆抗体揭示了硫酸化N-和O-聚糖在淋巴细胞归巢和免疫监视中的协同功能。
Cell surface glycans play pivotal roles in immune cell trafficking and immunity. Here we present an efficient method for generating anti-carbohydrate monoclonal antibodies (mAbs) using gene-targeted mice and describe critical glycans in lymphocyte homing. We immunized sulfotransferase GlcNAc6ST-1 and GlcNAc6ST-2 doubly deficient mice with sulfotransferase-overexpressing Chinese hamster ovary cells and generated two mAbs, termed S1 and S2. Both S1 and S2 bound high endothelial venules (HEVs) in the lymphoid organs of humans and wild-type mice, but not in those of doubly deficient mice. Glycan array analysis indicated that both S1 and S2 specifically bound 6-sulfo sialyl Lewis X and its defuco-sylated structure. Interestingly, S2 inhibited lymphocyte homing to peripheral lymph nodes by 95%, whereas S1 inhibited it by only 25%. S2 also significantly inhibited contact hypersensitivity responses and L-selectin-dependent leukocyte adhesion to HEVs. Immunohistochemical and Western blot analyses indicated that S1 preferentially bound sulfated O-glycans, whereas S2 bound both sulfated N- and O-glycans in HEVs. Furthermore, S2 strongly inhibited the N-glycan-dependent residual lymphocyte homing in mutant mice lacking sulfated O-glycans, indicating the importance of both sulfated N- and O-glycans in lymphocyte homing. Thus, the two mAbs generated by a novel method revealed the cooperative function of sulfated N- and O-glycans in lymphocyte homing and immune surveillance.