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.

The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals.

The DNA sequence of the two human mucin genes MUC2 and MUC6 have not been completely resolved due to the repetitive nature of their central exon coding for Proline, Threonine and Serine rich sequences. The exact nucleotide sequence of these exons has remained unknown for a long time due to limitations in traditional sequencing techniques. These are still very poorly covered in new whole genome sequencing projects with the corresponding protein sequences partly missing. We used a BAC clone containing both these genes and third generation sequencing technology, SMRT sequencing, to obtain the full-length contiguous MUC2 and MUC6 tandem repeat sequences. The new sequences span the entire repeat regions with good coverage revealing their length, variation in repeat sequences and their internal organization. The sequences obtained were used to compare with available sequences from whole genome sequencing projects indicating variation in number of repeats and their internal organization between individuals. The lack of these sequences has limited the association of genetic alterations with disease. The full sequences of these mucins will now allow such studies, which could be of importance for inflammatory bowel diseases for MUC2 and gastric ulcer diseases for MUC6 where deficient mucus protection is assumed to play an important role.

Searching the Evolutionary Origin of Epithelial Mucus Protein Components-Mucins and FCGBP.

The gel-forming mucins are large glycosylated proteins that are essential components of the mucus layers covering epithelial cells. Using novel methods of identifying mucins based on profile hidden Markov models, we have found a large number of such proteins in Metazoa, aiding in their classification and allowing evolutionary studies. Most vertebrates have 5-6 gel-forming mucin genes and the genomic arrangement of these genes is well conserved throughout vertebrates. An exception is the frog Xenopus tropicalis with an expanded repertoire of at least 26 mucins of this type. Furthermore, we found that the ovomucin protein, originally identified in chicken, is characteristic of reptiles, birds, and amphibians. Muc6 is absent in teleost fish, but we now show that it is present in animals such as ghost sharks, demonstrating an early origin in vertebrate evolution. Public RNA-Seq data were analyzed with respect to mucins in zebrafish, frog, and chicken, thus allowing comparison in regard of tissue and developmental specificity. Analyses of invertebrate proteins reveal that gel-forming-mucin type of proteins is widely distributed also in this group. Their presence in Cnidaria, Porifera, and in Ctenophora (comb jellies) shows that these proteins were present early in metazoan evolution. Finally, we examined the evolution of the FCGBP protein, abundant in mucus and related to gel-forming mucins in terms of structure and localization. We demonstrate that FCGBP, ubiquitous in vertebrates, has a conserved N-terminal domain. Interestingly, this domain is also present as an N-terminal sequence in a number of bacterial proteins.

Human trefoil factor 2 is a lectin that binds α-GlcNAc-capped mucin glycans with antibiotic activity against Helicobacter pylori.

Helicobacter pylori infection is the major cause of gastric cancer and remains an important health care challenge. The trefoil factor peptides are a family of small highly conserved proteins that are claimed to play essential roles in cytoprotection and epithelial repair within the gastrointestinal tract. H. pylori colocalizes with MUC5AC at the gastric surface epithelium, but not with MUC6 secreted in concert with TFF2 by deep gastric glands. Both components of the gastric gland secretome associate non-covalently and show increased expression upon H. pylori infection. Although blood group active O-glycans of the Lewis-type form the basis of H. pylori adhesion to the surface mucin layer and to epithelial cells, α1,4-GlcNAc-capped O-glycans on gastric mucins were proposed to inhibit H. pylori growth as a natural antibiotic. We show here that the gastric glycoform of TFF2 is a calcium-independent lectin, which binds with high specificity to O-linked α1,4-GlcNAc-capped hexasaccharides on human and porcine stomach mucin. The structural assignments of two hexasaccharide isomers and the binding active glycotope were based on mass spectrometry, linkage analysis, (1)H nuclear magnetic resonance spectroscopy, glycan inhibition, and lectin competition of TFF2-mucin binding. Neoglycolipids derived from the C3/C6-linked branches of the two isomers revealed highly specific TFF2 binding to the 6-linked trisaccharide in GlcNAcα1-4Galß1-4GlcNAcß1-6(Fucα1-2Galß1-3)GalNAc-ol(Structure 1). Supposedly, lectin TFF2 is involved in protection of gastric epithelia via a functional relationship to defense against H. pylori launched by antibiotic α1,4-GlcNAc-capped mucin glycans. Lectin-carbohydrate interaction may have also an impact on more general functional aspects of TFF members by mediating their binding to cell signaling 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.

Quantitative MUC5AC and MUC6 mucin estimations in gastric mucus by a least-squares minimization method.

We have determined the molar proportions of the MUC5AC and MUC6 mucus glycoproteins (mucins) in mucus from the normal and pathological human gastric antrum using a least-squares minimization analysis applied to amino acid compositions. We noted that the content of MUC5AC mucin in mucus from individuals without gastroduodenal disease was very high, suggesting that the integrity and barrier properties of the adherent gastric mucus layer are normally maintained by building-block structures formed from this mucin alone. We observed that the molar content of MUC6 mucin doubled (without significance) in mucus from patients with duodenal ulcer, and increased five times (with high significance) in mucus from patients with gastric ulcer, when compared with that in mucus from individuals without gastroduodenal disease.

Double-globular structure of porcine stomach mucin: a small-angle X-ray scattering study.

We present evidence from small-angle X-ray scattering synchrotron experiments that porcine stomach mucin (MUC6) contains a double-globular comb structure. Analysis of the amino acid sequence of the peptide comb backbone indicates that the globular structure is determined by both the charge and hydrophobicity of the amino acids and the placement of the short hydrophilic carbohydrate side chains (approximately 2.5 nm). The double-globular structure is, thus, due to a block copolymer type hydrophobic polyampholyte charge instability in contrast to the random copolymer instabilities observed previously with synthetic polyelectrolytes (particularly polystyrene sulfonates). Careful filtering was required to exclude multimonomer aggregates from the X-ray measurements. A double Guinier analysis ( R g approximately 26 nm) and a double power law fit are consistent with two globules per chain in low salt conditions. The average radius of the globules is approximately 10 nm in salt- free condition (double Guinier fit) and the average distance of intrachain separation of the globules is 48 nm. The addition of salt causes a significant decrease in the radius of gyration (14 nm 100 mM NaCl) of the chains and is attributed to the contraction of the glycosylated peptide spacer between the two globules (the globular size continues to be approximately 10 nm and the globule separation is then 18 nm). Without salt, the scaling of the semidilute mesh size (xi) as a function of the mucin concentration (c) is xi approximately c (-0.45)compared with xi approximately c (-0.28) in high salt conditions, highlighting the globular nature of the chains. In contrast, hydrophilic flexible polyelectrolytes have a stronger concentration dependence of xi when excess salt is added.