Anabel Gonzalez-Gil1, T August Li1, Ryan N Porell1, Steve M Fernandes1, Haley E Tarbox1, Hyun Sil Lee2, Kazuhiro Aoki3, Michael Tiemeyer3, Jean Kim4, Ronald L Schnaar5. 1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Md. 2. Department of Medicine, Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, Md. 3. Complex Carbohydrate Research Center, University of Georgia, Athens, Ga. 4. Department of Medicine, Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, Md; Department of Otolaryngology, Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Md. 5. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Md. Electronic address: schnaar@jhu.edu.
Abstract
BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.
BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.
Authors: Mats W Johansson; Elizabeth A Kelly; Christopher L Nguyen; Nizar N Jarjour; Bruce S Bochner Journal: Int Arch Allergy Immunol Date: 2018-06-07 Impact factor: 2.749
Authors: Catharina Steentoft; Sergey Y Vakhrushev; Hiren J Joshi; Yun Kong; Malene B Vester-Christensen; Katrine T-B G Schjoldager; Kirstine Lavrsen; Sally Dabelsteen; Nis B Pedersen; Lara Marcos-Silva; Ramneek Gupta; Eric Paul Bennett; Ulla Mandel; Søren Brunak; Hans H Wandall; Steven B Levery; Henrik Clausen Journal: EMBO J Date: 2013-04-12 Impact factor: 11.598
Authors: Sheena C Kerr; Jeanmarie R Gonzalez; Julia Schanin; Michael C Peters; Bart N Lambrecht; Emily C Brock; Annabelle Charbit; K M Ansel; Bradford A Youngblood; John V Fahy Journal: Clin Exp Allergy Date: 2020-07-08 Impact factor: 5.018
Authors: Anabel Gonzalez-Gil; Ryan N Porell; Steve M Fernandes; Eila Maenpaa; T August Li; Tong Li; Philip C Wong; Kazuhiro Aoki; Michael Tiemeyer; Zaikuan J Yu; Benjamin C Orsburn; Namandjé N Bumpus; Russell T Matthews; Ronald L Schnaar Journal: J Biol Chem Date: 2022-04-20 Impact factor: 5.486
Authors: Hyun Sil Lee; Anabel Gonzalez-Gil; Virginia Drake; T August Li; Ronald L Schnaar; Jean Kim Journal: Glycobiology Date: 2021-09-09 Impact factor: 4.313
Authors: Bradford A Youngblood; John Leung; Rustom Falahati; Jason Williams; Julia Schanin; Emily C Brock; Bhupinder Singh; Alan T Chang; Jeremy A O'Sullivan; Robert P Schleimer; Nenad Tomasevic; Christopher R Bebbington; Bruce S Bochner Journal: Cells Date: 2020-12-24 Impact factor: 6.600