Tissue distribution and expression dynamics of trefoil factor genes in the hydroid Hydractinia symbiolongicarpus.

Proteins of the trefoil factor family (TFF) participate in mucosal repair and are formed by single or tandemly repeated trefoil domains. TFFs have been extensively studied in mammals and amphibians, but they have not been functionally characterized in other animals. Here we report the identification of two genes expressed in the hydroid Hydractinia symbiolongicarpus, predicted to encode trefoil domain-containing peptides, one with four trefoil domains in tandem and the other one with a trefoil domain flanked by two ShKT domains. Differential expression analyses by qPCR after an immune challenge and an induced mechanical damage, reveal that the former gene (hysyTFF) had no significant changes in expression after the inductions. However, the latter (hysyTFF-like) was overexpressed after three hours post immune challenge and was downregulated after the first hour post epithelial damage. Immunoblot analyses using specific IgY antibodies revealed that hysyTFF is secreted as a high molecular weight complex. Finally, whole mount immunofluorescence assays showed that hysyTFF was predominantly expressed in the endoderm of stolons and polyps, and sparsely in the ectoderm of both polyps and larvae. Thus, the tissue distribution and expression dynamics of trefoil factor genes in H. symbiolongicarpus suggest that hysyTFF is part of an ancient mechanism of epithelial restitution, and the newly reported hysyTFF-like might act as an immune effector gene, perhaps encoding an antibacterial peptide.

Trefoil Factor 2 Expressed by the Murine Pancreatic Acinar Cells Is Required for the Development of Islets and for ß-Cell Function During Aging.

Exocrine-to-endocrine cross talk in the pancreas is crucial to maintain ß-cell function. However, the molecular mechanisms underlying this cross talk are largely undefined. Trefoil factor 2 (Tff2) is a secreted factor known to promote the proliferation of ß-cells in vitro, but its physiological role in vivo in the pancreas is unknown. Also, it remains unclear which pancreatic cell type expresses Tff2 protein. We therefore created a mouse model with a conditional knockout of Tff2 in the murine pancreas. We find that the Tff2 protein is preferentially expressed in acinar but not ductal or endocrine cells. Tff2 deficiency in the pancreas reduces ß-cell mass on embryonic day 16.5. However, homozygous mutant mice are born without a reduction of ß-cells and with acinar Tff3 compensation by day 7. When mice are aged to 1 year, both male and female homozygous and male heterozygous mutants develop impaired glucose tolerance without affected insulin sensitivity. Perifusion analysis reveals that the second phase of glucose-stimulated insulin secretion from islets is reduced in aged homozygous mutant compared with controls. Collectively, these results demonstrate a previously unknown role of Tff2 as an exocrine acinar cell-derived protein required for maintaining functional endocrine ß-cells in mice.

Salivary trefoil factor family peptide 3 (TFF3) and flow rate in persons with and without obstructive sleep apnea: A preliminary study.

OBJECTIVES: Obstructive sleep apnea (OSA) is one of the most common chronic diseases. Trefoil factor family 3 (TFF3) peptides are secreted by major and minor salivary glands and may be involved in the pathogenesis of OSA. This study aimed to evaluate salivary TFF3 and flow rate between those with and without OSA. MATERIAL AND METHODS: This was a prospective experimental study that enrolled patients with OSA and non-OSA. Total unstimulated saliva was collected, the salivary flow rate was measured, and the TFF3 level was analyzed by using a modified sandwich enzyme-linked immunosorbent assay. Baseline characteristics, TFF3 level, and salivary flow rate were compared between both groups. Factors associated with the TFF3 level and flow rate were computed by using multivariate linear regression analysis. RESULTS: Twenty-eight participants were recruited in the study: 20 patients with OSA (71.42%) and 8 non-OSA as control. The TFF3 and salivary flow rates between both groups of non-OSA versus OSA were comparable (TFF3 non-OSA 61.06 vs. OSA 96.00 ng/mg; p = .276 and flow rate non-OSA 0.40 vs. OSA 0.35 mL/min; p = .320). Factors associated with the TFF3 level were neck circumference with a negative coefficient of -16.419 (p = .042). For the salivary flow rate, only age was a significant factor with the coefficient of -0.013 (p = .044). CONCLUSIONS: TFF3 and salivary flow rate were comparable between patients with OSA and non-OSA. The factor associated with TFF3 level was neck circumference, while age was negatively associated with the salivary flow rate in patients with OSA.

Weifuchun alters tongue flora and decreases serum trefoil factor I levels in gastric intestinal metaplasia: A CONSORT-compliant article.

OBJECTIVE: To explore the molecular mechanisms of Weifuchun in the treatment of gastric intestinal metaplasia (GIM), we designed a preclinical pilot study to examine potential markers of disease progression based on alterations in the tongue flora. METHODS: Total 27 patients with GIM were treated with Weifuchun for 4 weeks and 26 volunteers as controls. Tongue coating bacteria were profiled using 16S rDNA high-throughput sequencing. Serum pepsinogen I and II levels were detected using the latex immunoturbidimetric assay. The levels of serum trefoil factor I was detected by ELISA. Microplate-based quantification was used to detect serum total bile acid (TBA). RESULTS: After treatment, the relative abundance of 4 dominant tongue coating genera (Granulicatella, Gemella, Lachnoanaerobaculum, and Neisseria) increased significantly wheras Alloprevotella, [Eubacterium] nodatum group, Prevotell, and Ruminococcaceae UCG-014 decreased (P < .05). The results showed that Alloprevotella and 3 rare tongue coating genera (Lautropia, Treponema 2, and Aliihoeflea) might be potential markers or target flora for the treatment of GIM. Kyoto encyclopedia of genes and genomes (KEGG) function prediction analysis showed that Weifuchun may regulate bile secretion and folate biosynthesis in patients with GIM. The level of serum trefoil factor I decreased significantly in response to Weifuchun treatment, which was consistent with the decrease in folate biosynthesis predicted by KEGG. CONCLUSION: Weifuchun may restore the balance of tongue flora by decreasing the levels of serum trefoil factor I, thereby providing a new way to measuring the underlying effectiveness and potential mechanisms of action of this traditional Chinese medicinal compound in the treatment of GIM.

Electroacupuncture ameliorates peptic ulcer disease in association with gastroduodenal microbiota modulation in mice.

Peptic ulcer disease (PUD) is a common disease and frequently encountered in the clinic. Accumulating evidence suggests that PUD is associated with the gastrointestinal microbiota. Electroacupuncture (EA) is an improved version of acupuncture, which can improve the clinical effect by increasing the stimulation and delivering appropriate electrical pulses to needles. This method has been widely used in the treatment of peptic ulcer disease. However, its effect on gastrointestinal microbiota remains unclear. Therefore, in the present study, the ameliorative effect of EA was evaluated on the gastroduodenal mucosa, and the regulatory effect of the gastroduodenal microbiota was assessed in PUD mice. A total of 48 male Kun Ming mice were randomly divided into the following groups: normal control group (NC), PUD model group (PUD), Shousanli group (LI10), and Zusanli group (ST36) (n=12). The mice in groups LI10 and ST36 were treated with EA at LI10 and ST36, respectively. This intervention was continued for 7 days. Subsequently, we evaluated the morphological changes in the gastric and duodenal mucosa, and specific indices were measured, including the contents of serum dopamine (DA), the trefoil factor (TFF), and the vasoactive intestinal peptide (VIP). In addition, the gastric and duodenal microbiota were assessed via 16S ribosomal DNA sequencing. The results indicated that EA at LI10 or ST36 significantly reduced the injury of the gastroduodenal mucosa in PUD mice. The gastric microbial community structure of the groups LI10 and ST36 was similar to that of the NC group following comparison with the microbial community structure of the PUD model group. Moreover, the abundance of Firmicutes in the stomach was decreased, whereas that of Bacteroidetes was increased, and the abundance of Firmicutes in the duodenum was decreased. Furthermore, the microbial diversity and richness of the gastric microbiota in group LI10 were also significantly increased, and the serum dopamine and trefoil factor levels in group ST36 were significantly increased. Therefore, it is suggested that EA ameliorating PUD is in association with improving the levels of DA and TFF and regulating the relative abundances of Firmicutes and Bacteroidetes in the gastric microbiota.

Pore-forming protein ßγ-CAT promptly responses to fasting with capacity to deliver macromolecular nutrients.

During animal fasting, the nutrient supply and metabolism switch from carbohydrates to a new reliance on the catabolism of energy-dense lipid stores. Assembled under tight regulation, ßγ-CAT (a complex of non-lens ßγ-crystallin and trefoil factor) is a pore-forming protein and trefoil factor complex identified in toad Bombina maxima. Here, we determined that this protein complex is a constitutive component in toad blood, that actively responds to the animal fasting. The protein complex was able to promote cellular albumin and albumin-bound fatty acid (FA) uptake in a variety of epithelial and endothelial cells, and the effects were attenuated by a macropinocytosis inhibitor. Endothelial cell-derived exosomes containing largely enriched albumin and FAs, called nutrisomes, were released in the presence of ßγ-CAT. These specific nutrient vesicles were readily taken up by starved myoblast cells to support their survival. The results uncovered that pore-forming protein ßγ-CAT is a fasting responsive element able to drive cell vesicular import and export of macromolecular nutrients.

The Association of Trefoil Factors with Gastric Cancer and Premalignant Lesions: A Cross-Sectional Population-Based Cohort Study.

BACKGROUND: A lack of research on the association of trefoil factors (TFF) with gastric cancer and premalignant lesions (PML) in the general population is an important obstacle to the application of TFFs for gastric cancer screening. We aimed to analyze the association of TFFs with gastric cancer and PMLs in a general population. METHODS: We evaluated 3,986 adults residing in Wuwei, China. We collected baseline characteristics and gastric cancer risk factors, including TFFs, endoscopic diagnosis, and pathologic information. Three logistic regression models were generated to analyze the association between TFFs and gastric cancer, as well as PMLs. Adjusted odds ratio (OR) and 95% confidence intervals (95% CI) were calculated to determine the strength of association. RESULTS: Compared with pepsinogen (PG) and anti-Helicobacter pylori immunoglobulin G antibody (Hp-IgG), TFFs had significant association with gastric cancer and PMLs after adjusting for biomarkers and risk factors (P < 0.05). The ORs (95% CI) for TFF1 (1.67; 1.27-2.20), TFF2 (2.66; 2.01-3.51), and TFF3 (1.32; 1.00-1.74) were larger than the ORs for PGI (0.79; 0.61-1.03), PGI/II (1.00; 0.76-1.31), and Hp-IgG (0.99; 0.73-1.35) in the gastric cancer group. In the intestinal metaplasia (IM) group, not only the TFF3 serum level was the highest, but also the OR (1.92; 1.64-2.25) was the highest. CONCLUSIONS: TFFs were associated with risk of gastric cancer and PMLs. IMPACT: Serum TFFs can improve the screening of high-risk populations for gastric cancer.

Trefoil Factor Family: A Troika for Lung Repair and Regeneration.

Tissue damage in the upper and lower airways caused by mechanical abrasion, noxious chemicals, or pathogenic organisms must be followed by rapid restorative processes; otherwise, persistent immunopathology and disease may ensue. This review will discuss evidence for the important role served by trefoil factor (TFF) family members in healthy and diseased airways of humans and rodents. Collectively, these peptides serve to both maintain and restore homeostasis through their regulation of the mucous layer and their control of cell motility, cell differentiation, and immune function in the upper and lower airways. We will also discuss important differences in which trefoil member tracks with homeostasis and disease between humans and mice, which poses a challenge for research in this area. Moreover, we discuss new evidence supporting newly identified receptor binding partners in the leucine-rich repeat and immunoglobulin-like domain-containing NoGo (LINGO) family in mediating the biological effects of TFF proteins in mouse models of epithelial repair and infection. Recent advances in our knowledge regarding TFF peptides suggest that they may be reasonable therapeutic targets in the treatment of upper and lower airway diseases of diverse etiologies. Further work understanding their role in airway homeostasis, repair, and inflammation will benefit from these newly uncovered receptor-ligand interactions.

The trefoil factor family 1 (TFF-1) and 3 (TFF-3) are upregulated in the saliva, gingival crevicular fluid and serum of periodontitis patients.

OBJECTIVE: This study aimed to investigate the levels of trefoil factor family (TFF)-1, TFF-3 and interleukin (IL)-1ß in gingival crevicular fluid (GCF), saliva and serum of patients with gingivitis, stage 3 periodontitis and healthy individuals. MATERIALS AND METHODS: A total of 100 individuals consisting of 25 periodontally healthy, 25 gingivitis and 50 stage 3 periodontitis, were enrolled in the study. Clinical periodontal examinations were recorded and GCF, saliva and serum samples were obtained. TFF-1, TFF-3 and IL-1ß were measured by ELISA. RESULTS: TFF-1 and TFF-3 levels in both GCF, saliva and serum were higher in periodontitis patients than healthy controls (p < .001) and gingivitis group (p < .01). The levels of these peptides in all biofluids were similar between gingivitis and healthy control groups (p > .05). GCF, saliva and serum IL-1ß levels were also higher in periodontitis patients than the controls (p < .01). Periodontitis patients had elevated GCF and saliva IL-ß levels than gingivitis group (p < .001). CONCLUSION: Elevated TFF-1 and TFF-3 levels both locally and systemically in periodontitis in parallel to increased IL-1ß levels might suggest that these peptides are involved in host response during the periodontal tissue destruction.

Salivary Trefoil Factor Family (TFF) Peptides and Their Roles in Oral and Esophageal Protection: Therapeutic Potential.

Human saliva is a complex body fluid with more than 3000 different identified proteins. Besides rheological and lubricating properties, saliva supports wound healing and acts as an antimicrobial barrier. TFF peptides are secreted from the mucous acini of the major and minor salivary glands and are typical constituents of normal saliva; TFF3 being the predominant peptide compared with TFF1 and TFF2. Only TFF3 is easily detectable by Western blotting. It occurs in two forms, a disulfide-linked homodimer (Mr: 13k) and a high-molecular-mass heterodimer with IgG Fc binding protein (FCGBP). TFF peptides are secretory lectins known for their protective effects in mucous epithelia; the TFF3 dimer probably has wound-healing properties due to its weak motogenic effect. There are multiple indications that FCGBP and TFF3-FCGBP play a key role in the innate immune defense of mucous epithelia. In addition, homodimeric TFF3 interacts in vitro with the salivary agglutinin DMBT1gp340. Here, the protective roles of TFF peptides, FCGBP, and DMBT1gp340 in saliva are discussed. TFF peptides are also used to reduce radiotherapy- or chemotherapy-induced oral mucositis. Thus, TFF peptides, FCGBP, and DMBT1gp340 are promising candidates for better formulations of artificial saliva, particularly improving wound healing and antimicrobial effects even in the esophagus.

An Aerolysin-like Pore-Forming Protein Complex Targets Viral Envelope to Inactivate Herpes Simplex Virus Type 1.

Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that ßγ-crystallin fused aerolysin-like protein and trefoil factor complex (ßγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. ßγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, ßγ-CAT formed ring-like oligomers of ∼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the ßγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that ßγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the ßγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of ßγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that ßγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.

Trefoil Factor Family (TFF) Peptides and Their Links to Inflammation: A Re-evaluation and New Medical Perspectives.

Trefoil factor family peptides (TFF1, TFF2, TFF3), together with mucins, are typical exocrine products of mucous epithelia. Here, they act as a gastric tumor suppressor (TFF1) or they play different roles in mucosal innate immune defense (TFF2, TFF3). Minute amounts are also secreted as endocrine, e.g., by the immune and central nervous systems. As a hallmark, TFF peptides have different lectin activities, best characterized for TFF2, but also TFF1. Pathologically, ectopic expression occurs during inflammation and in various tumors. In this review, the role of TFF peptides during inflammation is discussed on two levels. On the one hand, the expression of TFF1-3 is regulated by inflammatory signals in different ways (upstream links). On the other hand, TFF peptides influence inflammatory processes (downstream links). The latter are recognized best in various Tff-deficient mice, which have completely different phenotypes. In particular, TFF2 is secreted by myeloid cells (e.g., macrophages) and lymphocytes (e.g., memory T cells), where it modulates immune reactions triggering inflammation. As a new concept, in addition to lectin-triggered activation, a hypothetical lectin-triggered inhibition of glycosylated transmembrane receptors by TFF peptides is discussed. Thus, TFFs are promising players in the field of glycoimmunology, such as galectins and C-type lectins.

Animal secretory endolysosome channel discovery.

Secretory pore-forming proteins (PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs (af-PFPs) and trefoil factors (TFFs). As a toad af-PFP, BmALP1 can be reversibly regulated between active and inactive forms, with its paralog BmALP3 acting as a negative regulator. BmALP1 interacts with BmTFF3 to form a cellular active complex called ßγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of ßγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence, we hereby propose a secretory endolysosome channel (SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with ßγ-CAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms.

Trefoil Factor Family (TFF) Peptides and their Different Roles in the Mucosal Innate Immune Defense and More: An Update.

Mucous epithelia are protected by complex mucus barrier layers, which are part of the innate immune defense. Trefoil factor family peptides TFF1, TFF2, and TFF3 have lectin activities and are predominantly co-secreted together with mucins from these epithelia. TFF1 and TFF2 are mainly expressed in the gastric mucosa, whereas TFF3 is widely secreted from most mucous epithelia and their glands. TFF1 and TFF3 consist of a single TFF domain and an additional free 7th cysteine residue, whereas TFF2 contains two TFF domains. Systematic analyses of the molecular forms of TFFs gave new insights into their diverse molecular functions. TFF1 mainly exists as a monomer with an unusual free thiol group and only minor amounts form a disulfide-linked homodimer as well as heterodimers with gastrokine-2 and IgG-Fc-binding protein (FCGBP). TFF3 mainly forms a heterodimer with FCGBP in vivo, but also binds Deleted in Malignant Brain Tumors/gp340 (DMBT1gp340) in vitro. In contrast, TFF2 binds as a lectin to a conserved O-linked carbohydrate moiety of the mucin MUC6. Both FCGBP and DMBT1gp340 are secreted by most mucous epithelia and their glands and are involved in mucosal innate immunity. Thus, a new picture emerged pointing to functions of TFF3-FCGBP (and TFF1-FCGBP) for mucosal innate immune defense, e.g. supporting the clearing of the microorganisms. Such a function could be well be supported by DMBT1gp340. In contrast, the TFF2/MUC6 lectin complex probably physically stabilizes the inner adherent gastric mucus layer. Furthermore, there are indications that TFF3- FCGBP might also play a role in the blood vessels.

Urinary biomarkers as point-of-care tests for predicting progressive deterioration of kidney function in congenital anomalies of kidney and urinary tract: trefoil family factors (TFFs) as the emerging biomarkers.

BACKGROUND: Children with congenital anomalies of kidney and urinary tract (CAKUT) are at high risk of progressive deterioration of kidney function and further developing stage 5 chronic kidney disease (CKD 5), even after a successful surgery. This prospective study was designed to determine whether urinary biomarkers can predict progressive deterioration of kidney function in children with CAKUT. METHODS: The study included 50 consecutive children, aged < 14 years, who were diagnosed with congenital uropathies (PUV, VUR, and PUJO) and 20 age-matched controls. Examination of four urinary biomarkers, i.e., trefoil family factors (TFF) 1 and 3, neutrophil gelatinase-associated lipocalin (NGAL) and microalbuminuria (MALB) was done at the beginning of follow-up. Kidney function was assessed, at the beginning and after 12-months of follow-up, by technetium-99m diethylene triamine pentaacetic acid (DTPA) and technetium-99m dimercaptosuccinic acid (DMSA) scans. Progressive deterioration in the kidney function was defined as a fall in the GFR from ≥ 60 to < 60 ml/min/1.73 m2 on comparing the baseline and latest DTPA scans; and/or new-onset cortical scar/scars or increase in the size of previous scar/scars on serial DMSA scans. Group 1 and group 2 included children without and with progressive functional deterioration respectively. RESULTS: The median (IQR) age of children with CAKUT and controls was 3 (1.5-5) and 2.3 (1.2-3.6) years, respectively, and showed no significant difference (p = 0.29). Median concentrations of TFF1, TFF3, NGAL, and microalbumin in patients were 44.5, 176.5, 281.2, and 15.5 mcg/gCr, respectively, and were significantly elevated as compared to controls (p < 0.05). Children belonging to group 2 had significantly higher concentration of biomarkers as compared to those in group 1. TFF3 was found have the highest AUC (0.9198) on ROC curve for predicting progressive functional deterioration. CONCLUSION: Urinary TFFs, NGAL, and microalbumin significantly correlate with progressive deterioration of kidney function in children harboring CAKUT. TFF3, with the strongest prediction of functional deterioration, is an emerging peptide showing sufficient potential to be included in the biomarker panel. Graphical abstract.

Protective Effects of Wheat Peptides against Ethanol-Induced Gastric Mucosal Lesions in Rats: Vasodilation and Anti-Inflammation.

Alcohol consumption increases the risk of gastritis and gastric ulcer. Nutritional alternatives are considered for relieving the progression of gastric mucosal lesions instead of conventional drugs that produce side effects. This study was designed to evaluate the gastroprotective effects and investigate the defensive mechanisms of wheat peptides against ethanol-induced acute gastric mucosal injury in rats. Sixty male Sprague-Dawley rats were divided into six groups and orally treated with wheat peptides (0.1, 0.2, 0.4 g/kgbw) and omeprazole (20 mg/kgbw) for 4 weeks, following absolute ethanol administration for 1 h. Pretreatment with wheat peptides obviously enhanced the vasodilation of gastric mucosal blood vessels via improving the gastric mucosal blood flow and elevating the defensive factors nitric oxide (NO) and prostaglandin E2 (PGE2), and lowering the level of vasoconstrictor factor endothelin (ET)-1. Wheat peptides exhibited anti-inflammatory reaction through decreasing inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, and increasing trefoil factor 1 (TFF1) levels. Moreover, wheat peptides significantly down-regulated the expression of phosphorylated nuclear factor kappa-B (p-NF-κB) p65 proteins in the NF-κB signaling pathway. Altogether, wheat peptides protect gastric mucosa from ethanol-induced lesions in rats via improving the gastric microcirculation and inhibiting inflammation mediated by the NF-κB signaling transduction pathway.

Trefoil Factor Family (TFF) Peptides and Their Diverse Molecular Functions in Mucus Barrier Protection and More: Changing the Paradigm.

Trefoil factor family peptides (TFF1, TFF2, TFF3) are typically co-secreted together with mucins. Tff1 represents a gastric tumor suppressor gene in mice. TFFs are also synthesized in minute amounts in the immune and central nervous systems. In mucous epithelia, they support rapid repair by enhancing cell migration ("restitution") via their weak chemotactic and anti-apoptotic effects. For a long time, as a paradigm, this was considered as their major biological function. Within recent years, the formation of disulfide-linked heterodimers was documented for TFF1 and TFF3, e.g., with gastrokine-2 and IgG Fc binding protein (FCGBP). Furthermore, lectin activities were recognized as enabling binding to a lipopolysaccharide of Helicobacter pylori (TFF1, TFF3) or to a carbohydrate moiety of the mucin MUC6 (TFF2). Only recently, gastric TFF1 was demonstrated to occur predominantly in monomeric forms with an unusual free thiol group. Thus, a new picture emerged, pointing to diverse molecular functions for TFFs. Monomeric TFF1 might protect the gastric mucosa as a scavenger for extracellular reactive oxygen/nitrogen species. Whereas, the TFF2/MUC6 complex stabilizes the inner layer of the gastric mucus. In contrast, the TFF3-FCGBP heterodimer (and also TFF1-FCGBP) are likely part of the innate immune defense of mucous epithelia, preventing the infiltration of microorganisms.

Oligomerization of a symmetric ß-trefoil protein in response to folding nucleus perturbation.

Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN). In principle, more than one instance of the FN potentially enables an alternative folding pathway in the face of a subsequent deleterious mutation. We describe the targeted mutation of the carboxyl-terminal region of the (internally located) FN of the de novo designed purely-symmetric ß-trefoil protein Symfoil-4P. This mutation involves wholesale replacement of a repeating trefoil-fold motif with a "blade" motif from a ß-propeller protein, and postulated to trap that region of the Symfoil-4P FN in a nonproductive folding intermediate. The resulting protein (termed "Bladefoil") is shown to be cooperatively folding, but as a trimeric oligomer. The results illustrate how symmetric protein architectures have potentially diverse folding alternatives available to them, including oligomerization, when preferred pathways are perturbed.

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.

Odyssey of trefoil factors in cancer: Diagnostic and therapeutic implications.

Trefoil factors 1, 2, and 3 (TFFs) are a family of small secretory molecules involved in the protection and repair of the gastrointestinal tract (GI). TFFs maintain and restore epithelial structural integrity via transducing key signaling pathways for epithelial cell migration, proliferation, and invasion. In recent years, TFFs have emerged as key players in the pathogenesis of multiple diseases, especially cancer. Initially recognized as tumor suppressors, emerging evidence demonstrates their key role in tumor progression and metastasis, extending their actions beyond protection. However, to date, a comprehensive understanding of TFFs' mechanism of action in tumor initiation, progression and metastasis remains obscure. The present review discusses the structural, functional and mechanistic implications of all three TFF family members in tumor progression and metastasis. Also, we have garnered information from studies on their structure and expression status in different organs, along with lessons from their specific knockout in mouse models. In addition, we highlight the emerging potential of using TFFs as a biomarker to stratify tumors for better therapeutic intervention.