Correction: Chemical synthesis of human trefoil factor 1 (TFF1) and its homodimer provides novel insights into their mechanisms of action.

Correction for 'Chemical synthesis of human trefoil factor 1 (TFF1) and its homodimer provides novel insights into their mechanisms of action' by Nayara Braga Emidio et al., Chem. Commun., 2020, DOI: 10.1039/D0CC02321C.

Chemical synthesis of human trefoil factor 1 (TFF1) and its homodimer provides novel insights into their mechanisms of action.

TFF1 is a key peptide for gastrointestinal protection and repair. Its molecular mechanism of action remains poorly understood with synthetic intractability a recognised bottleneck. Here we describe the synthesis of TFF1 and its homodimer and their interactions with mucins and Helicobacter pylori. Synthetic access to TFF1 is an important milestone for probe and therapeutic development.

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.

The skin of the frog Xenopus laeevis is protected from microbial infections by a mucus barrier that contains frog integumentary mucins (FIM)-A.1, FIM-B.1, and FIM-C.1. These gel-forming mucins are synthesized in mucous glands consisting of ordinary mucous cells and one or more cone cells at the gland base. FIM-A.1 and FIM-C.1 are unique because their cysteine-rich domains belong to the trefoil factor family (TFF). Furthermore, FIM-A.1 is unusually short (about 400 amino acid residues). In contrast, FIM-B.1 contains cysteine-rich von Willebrand D (vWD) domains. Here, we separate skin extracts by the use of size exclusion chromatography and analyze the distribution of FIM-A.1 and FIM-C.1. Two mucin complexes were detected, i.e., a high-molecular-mass Complex I, which contains FIM-C.1 and little FIM-A.1, whereas Complex II is of lower molecular mass and contains the bulk of FIM-A.1. We purified FIM-A.1 by a combination of size-exclusion chromatography (SEC) and anion-exchange chromatography and performed first in vitro binding studies with radioactively labeled FIM-A.1. Binding of 125I-labeled FIM-A.1 to the high-molecular-mass Complex I was observed. We hypothesize that the presence of FIM-A.1 in Complex I is likely due to lectin interactions, e.g., with FIM-C.1, creating a complex mucus network.

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

TFF1 is a protective peptide of the Trefoil Factor Family (TFF), which is co-secreted with the mucin MUC5AC, gastrokine 2 (GKN2), and IgG Fc binding protein (FCGBP) from gastric surface mucous cells. Tff1-deficient mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas, indicating that Tff1 is a tumor suppressor. As a hallmark, TFF1 contains seven cysteine residues with three disulfide bonds stabilizing the conserved TFF domain. Here, we systematically investigated the molecular forms of TFF1 in the human gastric mucosa. TFF1 mainly occurs in an unusual monomeric form, but also as a homodimer. Furthermore, minor amounts of TFF1 form heterodimers with GKN2, FCGBP, and an unknown partner protein, respectively. TFF1 also binds to the mucin MUC6 in vitro, as shown by overlay assays with synthetic 125I-labeled TFF1 homodimer. The dominant presence of a monomeric form with a free thiol group at Cys-58 is in agreement with previous studies in Xenopus laevis and mouse. Cys-58 is likely highly reactive due to flanking acid residues (PPEEEC58EF) and might act as a scavenger for extracellular reactive oxygen/nitrogen species protecting the gastric mucosa from damage by oxidative stress, e.g., H2O2 generated by dual oxidase (DUOX).

The TFF Peptides xP1 and xP4 Appear in Distinctive Forms in the Xenopus laevis Gastric Mucosa: Indications for Different Protective Functions.

The gastric secretory trefoil factor family (TFF) peptides xP1 and xP4 are the Xenopus laevis orthologs of mammalian TFF1 and TFF2, respectively. The aim of this study was to analyze the molecular forms of xP1 and xP4 in the X. laevis gastric mucosa by FPLC. xP1 mainly occurred in a monomeric low-molecular-mass form and only a minor subset is associated with the mucus fraction. The occurrence of monomeric xP1 is unexpected because of its odd number of cysteine residues. Probably a conserved acidic residue flanking Cys55 allows monomeric secretion. Furthermore, Cys55 is probably post-translationally modified. For the first time, we hypothesize that the free thiol of monomeric xP1-and probably also its mammalian ortholog TFF1-could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. In contrast, xP4 mainly occurs in a high-molecular-mass form and is non-covalently bound to a mucin similarly as TFF2. In vitro binding studies with radioactively labeled porcine TFF2 even showed binding to X. laevis gastric mucin. Thus, xP4 is expected to bind as a lectin to an evolutionary conserved sugar epitope of the X. laevis ortholog of mucin MUC6 creating a tight mucus barrier. Taken together, xP1 and xP4 appear to have different gastric protective functions.

Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies.

Trefoil factor family 2 (TFF2) and the mucin MUC6 are co-secreted from human gastric and duodenal glands. TFF2 binds MUC6 as a lectin and is a constituent of the gastric mucus. Herein, we investigated human gastric extracts by FPLC and identified mainly high- but also low-molecular-mass forms of TFF2. From the high-molecular-mass forms, TFF2 can be completely released by boiling in SDS or by harsh denaturing extraction. The low-molecular-mass form representing monomeric TFF2 can be washed out in part from gastric mucosa specimens with buffer. Overlay assays with radioactively labeled TFF2 revealed binding to the mucin MUC6 and not MUC5AC. This binding is modulated by Ca2+ and can be blocked by the lectin GSA-II and the monoclonal antibody HIK1083. TFF2 binding was also inhibited by Me-ß-Gal, but not the α anomer. Thus, both the α1,4GlcNAc as well as the juxtaperipheral ß-galactoside residues of the characteristic GlcNAcα1→4Galß1→R moiety of human MUC6 are essential for TFF2 binding. Furthermore, there are major differences in the TFF2 binding characteristics when human is compared with the porcine system. Taken together, TFF2 appears to fulfill an important role in stabilizing the inner insoluble gastric mucus barrier layer, particularly by its binding to the mucin MUC6.

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

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 125 I-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→4Galß1→R moiety of MUC6. TFF2 binding was also inhibited in the presence of Me-ß-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.

Porcine gastric TFF2 is a mucus constituent and differs from pancreatic TFF2.

BACKGROUND/AIMS: TFF2 is a secretory peptide (106 amino acid residues) of the gastric mucosa, the porcine exocrine pancreas as well as immune cells and the CNS. It was the aim of this study to compare gastric and pancreatic TFF2. METHODS: TFF2 was purified from the porcine stomach and pancreas, respectively, by size-exclusion and anion-exchange chromatography and then analyzed by Western blots as well as mass spectrometry. RESULTS: Gastric and pancreatic TFF2 differed markedly, i.e. gastric TFF2 was exclusively associated with the high molecular mass mucus fraction, whereas pancreatic TFF2 appeared as a low molecular mass product. Unexpectedly, the latter predominantly formed a non-covalently linked homodimer resistant to boiling SDS. In contrast, gastric TFF2 is an integral mucus constituent predominantly binding to the mucin MUC6 in a non-covalent fashion. CONCLUSION: The non-covalent interaction of TFF2 with the mucin MUC6 is typical of a "link peptide" which is perfectly suited to assemble and stabilize the laminated structure of gastric mucus and to modulate its rheological properties.