Commercial Porcine Gastric Mucin Preparations, also Used as Artificial Saliva, are a Rich Source for the Lectin TFF2: In Vitro Binding Studies

Abstract

Mucous gels (mucus) cover internal body surfaces. The secretory mucins MUC5AC and MUC6 and the protective peptide TFF2 are characteristic constituents of gastric mucus; TFF2 is co‐secreted with MUC6. Herein, we investigated two commercial mucin preparations by FPLC and proteomics, because they are model systems for studying the rheology of gastric mucins. One preparation is also used as a saliva substitute, for example, after radiation therapy. We show that both preparations contain TFF2 (≈0.6 to 1.1 %, w/w). The majority of TFF2 is strongly bound noncovalently to mucin in a manner that is resistant to boiling in SDS. First overlay assays with 125I‐labeled porcine TFF2 revealed that mucin binding is modulated by Ca2+ and can be blocked by the lectin GSA‐II and the antibody HIK1083, both recognizing the peripheral GlcNAcα1→4Galβ1→R moiety of MUC6. TFF2 binding was also inhibited in the presence of Me‐β‐Gal but less so by the α anomer. TFF2 may play a role in the oligomerization and secretion of MUC6, the rheology of gastric mucus, and the adherence of gastric microbiota. TFF2 in artificial saliva may be of benefit. TFF2 might also interact with the sugar moiety of various receptors.

The Tumor Suppressor TFF1 Occurs in Different Forms and Interacts with Multiple Partners in the Human Gastric Mucus Barrier: Indications for Diverse Protective Functions

“…Additionally, anion-exchange chromatography was performed as reported previously [45,83] using a Resource Q6 column (Amersham Biosciences; salt gradient from 20 mM Tris-HCl pH 7.0 (buffer A) to 20 mM Tris-HCl pH 7.0 + 1 M NaCl (buffer B); flow rate: 4.0 mL/min, 1.0 mL fractions).…”

“…Labeling of synthetic TFF1 monomer (protected at Cys-58 with an acetamidomethyl group) and TFF1 homodimer with 125 I and overlay assays were performed analogous as described previously for TFF2 [83]. In brief, 6 µg TFF1 dimer (1 µg/µL) were labeled with 5 µL Na 125 I (100 mCi/mL, 2000 Ci/mmol; Hartmann Analytic GmbH, Braunschweig, Germany).…”

Trefoil Factor Family (TFF) Modules Are Characteristic Constituents of Separate Mucin Complexes in the Xenopus laevis Integumentary Mucus: In Vitro Binding Studies with FIM-A.1

“…Additionally, anion-exchange chromatography was performed as reported previously [33,41] using a Resource Q6 column (Amersham Biosciences; salt gradient from 20 mM Tris-HCl pH 7.0 (buffer A) to 20 mM Tris-HCl pH 7.0 + 1 M NaCl (buffer B); flow rate 6.0 mL /min, 1.0 mL fractions).…”

“…Western blot analysis after SDS-PAGE (electrophoretic transfer) or AgGE (capillary blot) was as reported [41]. All gels after non-reducing SDS-PAGE were subjected to post-in-gel reduction with 1% mercaptoethanol, as described previously [40].…”

“…Then, 500 µL of fraction B11 of the Resource Q6 column was desalted via a PD-10 desalting column (GE Healthcare Europe GmbH, Freiburg, Germany), and the three fractions positive for FIM-A.1 were concentrated with a Concentrator plus (Eppendorf, Hamburg, Germany) to a volume of 15 µL each. Labeling of FIM-A.1 with 125 I (iodogen method) and overlay assays with 125 I-labeled FIM-A.1 were analogous, as described for TFF2 previously [41]. In brief, mucin containing fractions after SEC were separated by AgGE, blotted onto nitrocellulose membranes, hybridized with 125 I-labeled FIM-A.1 (in 20 mM Tris-HCl pH 7.0, 2.5 mM CaCl 2 , 500 mM NaCl), and exposed to a film (autoradiography).…”