Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells—primarily eosinophils and mast cells—where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose–acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

中文翻译：

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唾液酸化硫酸角质素蛋白聚糖是人类呼吸道中的 Siglec-8 配体

人类 siglecs 是一个由 14 种唾液酸结合蛋白组成的家族，其中大部分在免疫细胞亚群上表达，在那里它们调节免疫反应。Siglec-8 在人类过敏性炎症细胞（主要是嗜酸性粒细胞和肥大细胞）上选择性表达，其中参与导致嗜酸性粒细胞凋亡并抑制肥大细胞介质释放。证据支持一种模型，其中人类嗜酸性粒细胞和肥大细胞与炎症靶组织上的 Siglec-8 唾液酸聚糖配体结合，以解决过敏性炎症并限制组织损伤。为了从人类呼吸道中鉴定与 Siglec-8 结合的唾液酸聚糖，从死后人类气管中提取的蛋白质通过尺寸排阻色谱和复合琼脂糖-丙烯酰胺凝胶电泳分离，通过 Siglec-8-Fc 印迹覆盖进行印迹和探测。确定了三种大小类别的 Siglec-8 配体：250 kDa、600 kDa 和 1 MDa，每一种都使用重组五聚体形式的 Siglec-8 通过亲和层析纯化。蛋白质组学质谱法将所有大小类别鉴定为蛋白聚糖聚集蛋白聚糖，这一发现经免疫印迹验证。聚糖阵列研究表明，Siglec-8 与合成聚糖结合，具有末端 Neu5Acα2-3(6-磺基)-Gal 行列式，这是聚集蛋白聚糖的硫酸角质素 (KS) 链上数量较少的末端。用唾液酸酶或角蛋白酶处理人气管提取物消除了 Siglec-8 结合，表明唾液酸化的 KS 链是 Siglec-8 结合决定簇。用角蛋白酶处理人气管组织切片也完全消除了 Siglec-8-Fc 的结合。最后，从人气管提取物中纯化的 Siglec-8 配体在体外诱导新鲜分离的人嗜酸性粒细胞凋亡增加。我们得出结论，唾液酸化 KS 蛋白聚糖是内源性人类呼吸道配体，可结合 Siglec-8 并可能调节过敏性炎症。