Nakamura Kyoko, Yamaji Toshiyuki, Crocker Paul R, Suzuki Akemi, Hashimoto Yasuhiro
Glyco-chain Functions Laboratory, Supra-biomolecular System Group, Frontier Research System, Institute of Physical and Chemical Research (RIKEN), Saitama, 351-0198, Japan.
Glycobiology. 2002 Mar;12(3):209-16. doi: 10.1093/glycob/12.3.209.
Sialoadhesin is a macrophage-restricted adhesion molecule that recognizes N-acetylneuraminylalpha2-3galactose structure. We prepared a multivalent neoglycoprotein probe carrying this oligosaccharide and characterized the binding activity of sialoadhesin on native rat macrophages. Macrophages from mesenteric and axillar lymph nodes exhibited 36-fold higher activity than those from the spleen. The K(d) values of the probe binding to macrophages of the two organs were indistinguishable (1-2 nM), whereas the B(max) value of lymph node macrophages was markedly higher than that of splenic macrophages. Western blot analysis revealed that the quantity of sialoadhesin present in lymph node macrophages was 25-fold higher than in splenic macrophages. High cell surface expression of sialoadhesin on lymph node macrophages was also shown by flow cytometry. To examine the "masking" of sialoadhesin by endogenous sialoglycoconjugates, we treated macrophages with sialidase before measuring the probe binding. After sialidase treatment, the binding activity of splenic macrophages increased fourfold, whereas that of lymph node macrophages did not increase. In conclusion, we have identified macrophages expressing high levels of unmasked sialoadhesin in lymph nodes. The unmasked forms on these macrophages are available for sialoadhesin-dependent adhesive functions, unlike the masked forms on the majority of splenic macrophages.
唾液酸黏附素是一种巨噬细胞限制性黏附分子，可识别N-乙酰神经氨酸α2-3半乳糖结构。我们制备了携带这种寡糖的多价新糖蛋白探针，并对唾液酸黏附素与天然大鼠巨噬细胞的结合活性进行了表征。来自肠系膜和腋窝淋巴结的巨噬细胞表现出比来自脾脏的巨噬细胞高36倍的活性。该探针与两个器官的巨噬细胞结合的K(d)值无显著差异（1-2 nM），而淋巴结巨噬细胞的B(max)值明显高于脾巨噬细胞。蛋白质印迹分析显示，淋巴结巨噬细胞中唾液酸黏附素的含量比脾巨噬细胞高25倍。流式细胞术也显示淋巴结巨噬细胞上唾液酸黏附素的细胞表面高表达。为了研究内源性唾液酸糖缀合物对唾液酸黏附素的“掩盖”作用，我们在测量探针结合之前用唾液酸酶处理巨噬细胞。唾液酸酶处理后，脾巨噬细胞的结合活性增加了四倍，而淋巴结巨噬细胞的结合活性没有增加。总之，我们已经鉴定出淋巴结中表达高水平未被掩盖的唾液酸黏附素的巨噬细胞。与大多数脾巨噬细胞上被掩盖的形式不同，这些巨噬细胞上未被掩盖的形式可用于唾液酸黏附素依赖性黏附功能。
