Solution structure of the disulfide-linked dimer of human intestinal trefoil factor (TFF3): the intermolecular orientation and interactions are markedly different from those of other dimeric trefoil proteins

The trefoil protein TFF3 forms a homodimer (via a disulfide linkage) that is thought to have increased biological activity over the monomer. The solution structure of the TFF3 dimer has been determined by NMR and compared with the structure of the TFF3 monomer and with other trefoil dimer structures (TFF1 and TFF2). The most significant structural differences between the trefoil domain in the monomer and dimer TFF3 are in the orientations of the N-terminal 3(10)-helix (residues 10-12) and in the presence in the dimer of an additional 3(10)-helix (residues 53-55) outside of the core region. The TFF3 dimer forms a more compact structure as compared with the TFF1 dimer where the two trefoil domains are connected by a flexible region with the monomer units being at variable distances from each other and in many different orientations. Although TFF2 is also a compact structure, the dispositions of its monomer units are very different from those of TFF3. The structural differences between the dimers result in the two putative receptor/ligand binding sites that remain solvent exposed in the dimeric structures having very different dispositions in the different dimers. Such differences have significant implications for the mechanism of action and functional specificity for the TFF class of proteins.     

Trefoil Factor 3 (TFF3) Is Regulated by Food Intake,Improves Glucose Tolerance and Induces Mucinous Metaplasia

Trefoil factor 3 (TFF3), also called intestinal trefoil factor or Itf, is a 59 amino acid peptide found as a homodimer predominantly along the gastrointestinal tract and in serum. TFF3 expression is elevated during gastrointestinal adenoma progression and has been shown to promote mucosal wound healing. Here we show that in contrast to other trefoil factor family members, TFF1 and TFF2, TFF3 is highly expressed in mouse duodenum, jejunum and ileum and that its expression is regulated by food intake. Overexpression of TFF3 using a recombinant adeno-associated virus (AAV) vector, or daily administration of recombinant TFF3 protein in vivo improved glucose tolerance in a diet-induced obesity mouse model. Body weight, fasting insulin, triglyceride, cholesterol and leptin levels were not affected by TFF3 treatment. Induction of mucinous metaplasia was observed in mice with AAV-mediated TFF3 overexpression, however, no such adverse histological effect was seen after the administration of recombinant TFF3 protein. Altogether these results suggest that the therapeutic potential of targeting TFF3 to treat T2D may be limited.     

三叶因子:从实验室研究到临床医学

三叶因子（trefoil factor, TFF）家族是具有一个或多个三叶因子结构域的蛋白质多肽,在进化上具有高度的保守性,具有耐热、耐酶消化的理化特性。哺乳动物的TFF有三个成员(TFF1、TFF2和TFF3)。黏膜组织,如胃肠道和呼吸道黏膜等是TFF的主要合成场所。生理条件下,TFF的分布具有组织专一性,具有黏膜保护和创伤修复作用。TFF在肿瘤组织中广泛表达,在肿瘤的发生、发展、侵袭、转移过程中发挥着重要作用,可能是癌基因在对不同刺激做出反应时的共有递质。TFF生物学功能存在一个复杂的调控过程,单链TFF可通过膜受体而激活相关信号通路,TFF也可以通过与其它蛋白质的结合而协同发挥作用。TFF在临床医学领域主要运用在黏膜保护、黏膜损伤相关疾病的预防和治疗及肿瘤病理的诊断与干预等方面。TFF的作用机制和功能行使相关信号通路仍然是目前研究的重点,TFF与βγ-晶状体蛋白天然复合物的发现有助于对TFF的进一步认识和深入研究。     

Injected TFF1 and TFF3 bind to TFF2-immunoreactive cells in the gastrointestinal tract in rats

Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are co-secreted with mucus in most organ systems and are believed to interact with mucins to produce high-viscosity, stable gel complexes. We have previously demonstrated that cells in the GI tract possess binding sites to TFF2 and that injected TFF2 ends up in the mucus layer. In the present study, tissue binding and metabolism of parenterally administered human TFF1 and TFF3 in rats were described and compared to the immunohistochemical localization of the TFF peptides. 125I-TFF1 monomer and 125I-TFF3 mono- and dimer were given intravenously to female Wistar rats. The tissue distribution was assessed by gamma counting of organ samples and by autoradiography of histological sections. The degradation of 125I-TFF3 was studied by means of trichloracetic acid (TCA) precipitation and the saturability of the binding by administration of excess unlabelled peptide. The TFF peptides were localized in histologic sections from the GI tract by immunohistochemistry. Injected TFF3 dimer (12%) was taken up by the GI tract. At autoradiography, grains were localized to the same cells that were immunoreactive to TFF2. The binding could be displaced by excess TFF3. Similar binding was observed for the TFF1 and TFF3 monomers apart from binding in the stomach, where the uptake was only 15% in comparison to the dimer. There was no specific binding outside the GI tract and no binding to TFF1 or TFF3 immunoreactive cells. In conclusion, the TFF2-binding cells in the gastrointestinal tract seem to have basolateral, receptor-like activity to all three TFF peptides. The mucous neck cells of the stomach predominantly take up TFFs with two trefoil domains, indicating a different receptor-like activity in the stomach compared to the rest of the GI tract.     

Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva

Background

Human trefoil factor (TFF) peptides consist of three members: TFF1, TFF2 and TFF3. TFF3 is the most abundant TFF peptide in saliva. TFF3 homodimer was suggested to be involved in apoptosis inhibition and malignancy. Determination of TFF3 homodimer expression profiles in saliva may lead to new information about oral biology and diseases. The objective of this study was to generate monoclonal antibodies (mAbs) against TFF3 and apply the produced mAbs for the establishment of ELISA for quantification of dimeric TFF3 in saliva.

Results

With our modified hybridoma technique, three hybridoma clones producing anti-TFF3 mAbs having IgG isotype were generated. The mAbs were specific for TFF3 with no cross-reactivity to other TFFs. Using the generated mAbs, a modified-sandwich ELISA with high sensitivity for the quantification of dimeric TFF3 in saliva was developed. Using this ELISA, the amount of dimeric TFF3 in saliva could be measured.

Conclusions

A modified-sandwich ELISA for the quantification of TFF3 dimeric form was established. The established ELISA will be a valuable tool for facilitating the investigation of the physiological roles and the diagnostic values of TFF3 in oral diseases. The concept of this modified-sandwich ELISA may be applied for the determination of other homodimeric peptides of interest.

     

The trefoil factor interacting protein TFIZ1 binds the trefoil protein TFF1 preferentially in normal gastric mucosal cells but the co-expression of these proteins is deregulated in gastric cancer

The gastric tumour suppressor trefoil protein TFF1 is present as a covalently bound heterodimer with a previously uncharacterised protein, TFIZ1, in normal human gastric mucosa. The purpose of this research was firstly to examine the molecular forms of TFIZ1 present, secondly to determine if TFIZ1 binds other proteins apart form TFF1 in vivo, thirdly to investigate if TFIZ1 and TFF1 are co-regulated in normal gastric mucosa and fourthly to determine if their co-regulation is maintained or disrupted in gastric cancer. We demonstrate that almost all human TFIZ1 is present as a heterodimer with TFF1 and that TFIZ1 is not bound to either of the other two trefoil proteins, TFF2 and TFF3. TFIZ1 and TFF1 are co-expressed by the surface mucus secretory cells throughout the stomach and the molecular forms of each protein are affected by the relative abundance of the other. TFIZ1 expression is lost consistently, early and permanently in gastric tumour cells. In contrast, TFF1 is sometimes expressed in the absence of TFIZ1 in gastric cancer cells and this expression is associated with metastasis (lymph node involvement: p=0.007). In conclusion, formation of the heterodimer between TFIZ1 and TFF1 is a specific interaction that occurs uniquely in the mucus secretory cells of the stomach, co-expression of the two proteins is disrupted in gastric cancer and expression of TFF1 in the absence of TFIZ1 is associated with a more invasive and metastatic phenotype. This indicates that TFF1 expression in the absence of TFIZ1 expression has potentially deleterious consequences in gastric cancer.     

Development of a two-site ELISA assay for the dimeric form of human TFF1

TFF1 is one of three human trefoil proteins expressed principally in the gastrointestinal tract in normal tissues. TFF1 protects the gastric mucosa against damage as a result of its ability to facilitate reconstitution of damaged gastric mucosa and its involvement in the secretion and structure of gastric mucus. The most biologically active molecular form in cell culture and animal models tested is a dimer formed by a disulfide bond between two cysteine residues close to the C terminus of the protein. We have therefore developed an assay for this form of TFF1 which should facilitate its measurement in biological samples.     

Interaction between TFF1, a gastric tumor suppressor trefoil protein, and TFIZ1, a brichos domain-containing protein with homology to SP-C

TFF1 is a gastric tumor suppressor that protects gastric epithelial cells from damage but can promote invasive properties of tumor cells. Antibodies were raised against correctly folded TFF1 protein. These showed that the 6.67 kDa secreted trefoil protein is present as an approximately 25 kDa complex in normal human gastric mucosa. The TFF1 complex was immunopurified from human gastric mucosa and shown to comprise two proteins joined by a disulfide bond. Both were identified by amino-terminal sequencing and MALDI TOF mass spectrometry. The TFF1 protein partner is a previously unknown protein that we have called TFIZ1 for trefoil factor interactions(z) 1. TFIZ1 is expressed and secreted in normal gastric mucosa. TFIZ1 mRNA was cloned from gastric mucosa and sequenced. TFIZ1 is an 18.31 kDa protein and contains an approximately 100 amino acid brichos domain and homology with smart00019.10, SF_P. This is the first demonstration that a member of the trefoil factor family of proteins is bound covalently to a brichos domain-containing protein. The apparent molecular mass of the TFF1:TFIZ1 heterodimer is remarkably close to the theoretical molecular mass of 24.98 kDa. In conclusion, the heterodimer comprises one molecule each of TFF1 and TFIZ1, and the disulfide bond between TFF1 and TFIZ1 is the most important factor stabilizing the heterodimer.     

Expression of trefoil peptides in the subtypes of intestinal metaplasia

We studied the expression of trefoil peptides in the different types of intestinal metaplasia of the stomach. Endoscopic biopsy was performed in 132 patients with dyspepsia. Intestinal metaplasia subtype was classified according to the pattern of alcian blue/PAS staining and high iron diamine staining. Expression of trefoil peptides was measured by immunohistochemistry. TFF1 and TFF3 were mainly expressed in goblet cells and TFF2 in columnar cells in all the types of intestinal metaplasia. There was a gradual decrease of TFF1 and TFF3, and increase of TFF2, during the progression of intestinal metaplasia from type I to type III via the type II intermediate.     

Alterations in the composition of the supramucosal defense barrier in relation to disease severity of ulcerative colitis.

Mucin glycoproteins and trefoil peptides play an important role in protection and repair of the gastrointestinal epithelium. This study investigates alterations in mucin and trefoil peptide gene expression and product localization in ulcerative colitis (UC). Product localization and message expression of mucin MUC1 to 6 and trefoil peptide TFF1 to 3 genes was analyzed in rectosigmoid tissue from a cohort of patients with active UC and compared with that of normal colorectal mucosa. MUC1 expression was upregulated in severe UC at the site of rupture of crypt abscesses. Reduction in MUC2 expression occurred in UC adjacent to ulceration. No alteration in MUC3 or MUC4 gene expression was detectable in UC compared with normal colorectal mucosa. No ectopic expression of MUC5AC, MUC5B, or MUC6 was identified in UC. Ectopic TFF1 expression was identified in tissues eliciting histological features of severe disease. Decreased TFF3 localization was demonstrated in UC tissues, but no TFF2 expression was detected in any colorectal specimens. Subtle alterations in composition of the supramucosal defense barrier exist in UC and vary in relation to clinical severity of disease. There is upregulation in mucin MUC1 at crypt abscesses and neo-expression of TFF1 trefoil peptide in severe disease.     

The closely related estrogen-regulated trefoil proteins TFF1 and TFF3 have markedly different hydrodynamic properties,overall charge,and distribution of surface charge

The human trefoil proteins TFF1 and TFF3 are expressed predominantly in the gastrointestinal tract. They are also expressed and regulated by estrogens in malignant breast epithelial cells. TFF1 and TFF3 are small cysteine-rich acidic secreted proteins of 60 and 59 amino acids with similar isoelectric points of 4.75 and 3.94, respectively. Each contains one trefoil domain that is characterized by several conserved features including six cysteine residues with conserved spacing. TFF1 and TFF3 form intermolecular disulfide bonds via an extra-trefoil domain cysteine residue and are present in vivo as monomers and homodimers and as complexes with other proteins. The TFF1 dimer is more active than the TFF1 monomer. In the present study the hydrodynamic and charge properties of TFF1 and TFF3 monomers and homodimers have been compared and shown to differ markedly. Notably, TFF1 is significantly more asymmetric than TFF3 (frictional coefficients 1.25 and 1.12, respectively, p < 0.001), and homodimerization of TFF1 results in a greater increase in asymmetry than for TFF3. The overall charges of TFF1 and TFF3 are very different at neutral pH. Titration curves predicted significant differences in charge across a wide pH range that agreed well with experimental data. The locations of charged amino acids in the primary sequences and in the tertiary structures of TFF1 and TFF3 were examined. This revealed interesting divergence in both the distribution and local topology of charged amino acid side chains. The significant differences between the shape, size, and surface charge of these two closely related molecules may account for their divergent biological activities.     

Co-localization of TFF3 peptide and oxytocin in the human hypothalamus.

TFF-peptides (formerly P domain peptides, trefoil factors) are typical secretory products of many mucous epithelial cells. TFF3 is also synthesized in the hypothalamus and has anxiolytic or anxiogenic activities when injected into the rat amygdala. Here we show by immunohistochemistry that TFF3 is localized to a distinct population of neurons of the human hypothalamic paraventricular and supraoptic nuclei. Generally, TFF3-positive cells are co-localized in oxytocin-producing cells and not in vasopressin-producing cells. Relatively large amounts of TFF3-but not TFF1 and TFF2-are present in the posterior lobe of the human pituitary, where it is probably released into the bloodstream. Furthermore, TFF3 was also detectable in human postmortem cerebrospinal fluid.     

Expression and localization of trefoil factor family genes in rat submandibular glands

The trefoil factor (TFF) family, which comprises TFF1, TFF2 and TFF3, plays an essential role in epithelial regeneration within the gastrointestinal tract. All three TFFs are present in human saliva; TFF3 is the predominant trefoil peptide. Little is known about the expression and tissue distribution of TFFs in rats, which are commonly used as a model system for human studies. We investigated the localization of the TFF genes that encode secretory peptides in rat submandibular glands (SMG). All three TFFs were expressed in rat SMG, although their location varied. Substantial amounts of TFF1 were detected only in the cytoplasm of epithelial cells in the SMG granular convoluted tubules (GCT), while TFF2 and TFF3 were widely distributed in the cytoplasm of epithelial cells of intercalated ducts (ID), striated ducts (SD) and interlobular ducts (ILD). The three TFFs also were detected especially in the lumens of the SD and ILD. Semi-quantitative RT-PCR and in situ hybridization experiments confirmed TFF1, TFF2 and TFF3 mRNA expressions in the SMG. Greater expression of TFF peptides and mRNA was observed in male rats than in females. The broad expression of TFFs in rat SMG cells and lumens suggests that TFFs function in this organ by their secretion into the duct lumens. We also found differences in TFF expression profiles between rat and human SMG; therefore, caution should be exercised when using rats as a model for human TFF studies.     

Expression of Trefoil Factor 1 in the Developing and Adult Rat Ventral Mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson’s disease.     

TFF peptides in the human false vocal folds of the larynx

TFF peptides (formerly P domain peptides, trefoil factors) are typical secretory products of mucin-producing cells and are thought to influence the rheological properties of mucous gels. We investigated the localization of these peptides in the human false vocal folds of the larynx, also known as the ventricular folds or vestibular folds. An analysis of TFF peptide mRNA by RT-PCR and TFF protein by Western blot detected TFF1 and TFF3, but not TFF2. Immunohistochemistry revealed TFF1 to be associated with the secretory product of goblet cells and mucous parts of subepithelial seromucous glands. TFF3 occurred in columnar epithelial cells of the mucosa and in serous cells and excretory duct cells of seromucous glands. These peptides may play a role in the rheological function of mucus secreted onto the true vocal folds and are thus important constituents of vocal production.     

Ultrastructural co-localization of TFF3-peptide and oxytocin in the neural lobe of the porcine pituitary

TFF-peptides (formerly P-domain peptides, trefoil factors) are typical secretory products of many mucous epithelial cells. TFF3 is also synthesized in oxytocinergic neurons in the paraventricular and supraoptic nuclei of the human hypothalamus. Here, TFF3 and oxytocin are shown to be co-localized within the same secretory vesicles in the neural (posterior) lobe of the procine pituitary by means of immunoelectron microscopy. Relatively large amounts of TFF3, but not TFF1 and TFF2, are present in the neural lobe of the porcine pituitary, where it is probably released into the bloodstream.     

1H NMR-based determination of the secondary structure of porcine pancreatic spasmolytic polypeptide: one of a new family of "trefoil" motif containing cell growth factors.

Two-dimensional 1H NMR spectroscopy has been used to obtain comprehensive sequence-specific resonance assignments for the putative cell growth factor porcine pancreatic spasmolytic polypeptide, which is a 106-residue protein containing two "trefoil" domains. The patterns of sequential (i,i+l), medium-range (i,i less than 5), and long-range NH to NH, alpha CH to NH, and alpha CH to alpha CH nuclear Overhauser effects clearly show that the protein's two trefoil domains adopt essentially the same secondary structure in solution. The main feature of each domain is a seven-residue helix followed by a short antiparallel beta-sheet formed from two strands of four amino acids each. This is a novel supersecondary structure, which clearly identifies the trefoil motif as a new class of growth factor associated module, distinct from other types of highly disulfide cross-linked domains, such as those found in epidermal growth factor and insulin-like growth factor I.     

Synthesis and localization of the mucin-associated TFF-peptides in the human uterus

TFF-peptides (formerly P-domain peptides, trefoil factors) are typical secretory products of mucin-producing cells and are thought to influence the rheological properties of mucous gels. Here, the localization of these peptides in the human uterus was investigated. An analysis of TFF-peptides mRNA by the polymerase chain reaction revealed TFF3 mainly in the endocervix and smaller amounts in the endometrium. TFF1 and TFF2 mRNA was detectable occasionally in the endocervix and very rarely in the endometrium. Western blot analysis detected only TFF3 in tissue extracts of the endocervix and as a constituent of human cervical mucus. Immunofluorescence localized TFF3 in the surface epithelium of the endocervix and in gland-like structures of the cervical epithelium.