Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, United States.
Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, IL 60607, United States.
Glycobiology. 2024 Aug 30;34(10). doi: 10.1093/glycob/cwae065.
Human sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed on subsets of immune cells. Siglec-8 is an immune inhibitory Siglec on eosinophils and mast cells, which are effectors in allergic disorders including eosinophilic esophagitis. Inhibition occurs when Siglec-8 is crosslinked by multivalent Siglec ligands in target tissues. Previously we discovered a high-affinity Siglec-8 sialoglycan ligand on human airways composed of terminally sialylated keratan sulfate chains carried on a single protein, DMBT1. Here we extend that approach to another allergic inflammatory target tissue, human esophagus. Lectin overlay histochemistry revealed that Siglec-8 ligands are expressed predominantly by esophageal submucosal glands, and are densely packed in submucosal ducts leading to the lumen. Expression is tissue-specific; esophageal glands express Siglec-8 ligand whereas nearby gastric glands do not. Extraction and resolution by gel electrophoresis revealed a single predominant human esophageal Siglec-8 ligand migrating at >2 MDa. Purification by size exclusion and affinity chromatography, followed by proteomic mass spectrometry, revealed the protein carrier to be MUC5B. Whereas all human esophageal submucosal cells express MUC5B, only a portion convert it to Siglec-8 ligand by adding terminally sialylated keratan sulfate chains. We refer to this as MUC5B S8L. Material from the esophageal lumen of live subjects revealed MUC5B S8L species ranging from ~1-4 MDa. We conclude that MUC5B in the human esophagus is a protein canvas on which Siglec-8 binding sialylated keratan sulfate chains are post-translationally added. These data expand understanding of Siglec-8 ligands and may help us understand their roles in allergic immune regulation.
人类唾液酸结合免疫球蛋白样凝集素（Siglecs）表达于免疫细胞亚群上。Siglec-8 是嗜酸性粒细胞和肥大细胞上的免疫抑制型 Siglec，在包括嗜酸性食管炎在内的过敏疾病中发挥效应器作用。当 Siglec-8 被靶组织中的多价 Siglec 配体交联时，就会发生抑制。此前，我们在人类气道中发现了一种高亲和力的 Siglec-8 糖基化配体，由单个蛋白 DMBT1 携带的末端唾液酸化角蛋白硫酸盐链组成。在此，我们将该方法扩展到另一种过敏炎症靶组织，即人类食管。凝集素覆盖组织化学显示，Siglec-8 配体主要表达于食管黏膜下腺，并且在通向管腔的黏膜下导管中密集排列。表达具有组织特异性；食管腺表达 Siglec-8 配体，而附近的胃腺则不表达。通过凝胶电泳进行提取和分辨率分析显示，单一的主要人食管 Siglec-8 配体迁移率大于 2MDa。通过大小排阻和亲和层析进行纯化，随后进行蛋白质组学质谱分析，揭示了蛋白载体为 MUC5B。尽管所有人类食管黏膜下细胞都表达 MUC5B，但只有一部分通过添加末端唾液酸化的角蛋白硫酸盐链将其转化为 Siglec-8 配体。我们将其称为 MUC5B S8L。来自活体受试者食管腔的材料显示，MUC5B S8L 种类范围为 1-4MDa。我们得出结论，人类食管中的 MUC5B 是一个蛋白质画布，其上可以通过翻译后添加与 Siglec-8 结合的唾液酸化角蛋白硫酸盐链。这些数据扩展了对 Siglec-8 配体的理解，并可能有助于我们理解它们在过敏免疫调节中的作用。
