Localization of TFF3 peptide to porcine conjunctival goblet cells

TFF-peptides (formerly P-domain peptides, trefoil factors) form a new family of mucin-associated peptides mainly in the gastrointestinal tract. TFF3 is a typical secretory product of intestinal goblet cells and occurs also in the respiratory tract. Here, polyclonal antisera specific for TFF3 were used in Western blot analysis and immunofluorescence to determine the presence and distribution of TFF3 in the porcine conjunctiva, which is the primary source for ocular mucins. Significant accumulation of TFF3 was detected in conjunctival goblet cells but not in the lacrimal glands. This peptide, together with ocular mucins, may play a role in the rheological function of the tear film.     

Characterization of the in vivo forms of lacrimal-specific proline-rich proteins in human tear fluid

The tear film is complex and is rich in both peptides and proteins. Physiological factors have been shown to alter the balance of the protein components in the tear film, however, little is known of the precise stimuli that initiate these changes, or their nature and extent. Attention has been directed at the role of tear proteins in the protection of the external ocular surface, and their potential role in the pathogenesis of inflammatory and autoimmune diseases, but few lacrimal-specific proteins have been identified and demonstrated to offer a protective function at the ocular surface. The biological importance of proline-rich proteins is uncertain, although there is some evidence to indicate a potential antimicrobial function for these proteins in saliva. Despite the detection of mRNA for proline-rich proteins in lacrimal gland, the translated protein product has not been detected in tear fluid. In this study we investigate the presence of proline-rich proteins in the tear film. Human reflex tear fluid was examined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry directly, and following size exclusion high performance liquid chromatography. This revealed significant levels of a truncated form of lacrimal proline-rich protein, and a series of peptides derived the C-terminus of this protein. None of these had previously been identified in tear. Our study highlights the dangers inherent in proteomic strategies that assign an identity to a protein based on limited coverage of tryptic peptides.     

Fresh and cryopreserved amniotic membrane secrete the trefoil factor family peptide 3 that is well known to promote wound healing

Amniotic membrane (AM) is often used for the treatment of ocular surface ulcerations and other corneal defects. Trefoil factor family (TFF) peptide 3 is produced by conjunctival goblet cells, participates in tear film physiology and has also been shown to be involved in ocular surface restitution after corneal injury. In the present study, we questioned whether AM also might be a source of TFF3 and if yes whether the secretion rate of TFF3 is changed by proinflammatory cytokines or by cryoconservation of AM. By means of RT-PCR, the mRNA expression of all three known TFF peptides could be detected in AM. Immunohistochemistry on paraffin-embedded sections localized TFF3 protein and also TFF2 in AM cells and Western blot analysis revealed TFF3 protein in AM. Stimulation experiments with proinflammatory cytokines and subsequent TFF3 ELISA measurements revealed that the secretion rate of fresh or cryoconserved AM was not significantly changed. The results indicate that TFF peptides are produced by AM. TFF3 may contribute to ocular surface wound healing after AM transplantation, but its production by AM is not further inducible by proinflammatory stimuli. Cryopreservation has no effect on the secretion rate of TFF3 supporting the use of cryopreserved AM for transplantation.     

Trefoil peptides promote restitution of wounded corneal epithelial cells

The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.     

Elastohydrodynamics of the Eyelid Wiper

This paper presents an elastohydrodynamic model of the human eyelid wiper. Standard lubrication theory is applied to the fluid layer between the eyelid wiper and ocular surface. The role of the lubrication film is to reduce the shear stresses by preventing solid to solid contact between the eyelid wiper and ocular surface. For the lubrication film to be effective, it is required that the orientation of the eyelid wiper changes between the opening and closing phases of a blink. In order to model this, the hydrodynamic model is coupled with an elastic mattress model for the soft tissue of the eyelid wiper and ocular surface. This leads to a one-dimensional non-linear partial differential equation governing the fluid pressure in the lubrication film. In order to solve the differential equation, a loading condition or constraint equation must be specified. The resulting system is then solved numerically. The model allows predictions of the tear film flux from under the upper eyelid, as well as normal and shear stresses acting on the ocular surface. These factors are important in relation to dry eye syndrome, deformation of the cornea and contact lens design. It is found that the pressure and shear stress under the eyelid act across a length of approximately 0.1 mm which is consistent with clinical observations. It order to achieve a flow of tears from under the upper eyelid during a blink, the model requires that the normal force the eyelid applies to the ocular surface during the closing phase of the blink is significantly higher than during the opening phase of the blink. Electronic Supplementary Material The online version of this article () contains supplementary material, which is available to authorized users.     

Identification of hemopexin in tear film

Human tear fluid is a complex mixture of aqueous lipids, proteins, enzymes, and other biochemical and cellular elements. By conventional comparative proteomic approaches, we investigated the proteome in human tear fluid and compared the tear protein profile of normal control subjects with that of patients suffering from the ocular inflammatory disease vernal keratoconjunctivitis (VKC). Collected tear samples were directed to two-dimensional polyacrylamide gel electrophoresis protein separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide identification. Six differentially expressed proteins—interleukin 4, phospholipase A2, albumin, lactoferrin, hemopexin, and lipocalin—were displayed. Hemopexin had not been reported previously in tear film. Enzyme-linked immunosorbent assay confirmed that hemopexin concentrations were significantly higher in VKC tear samples and increased with disease stages. The results implied clinical interest of hemopexin in the tear proteome and eye diseases.     

The Effect of the Lipid Layer on Tear Film Behaviour

This paper investigates the effect of surfactants during tear film deposition and subsequent thinning. The surfactants occur naturally on the surface of the tear film in the form of a lipid layer. A lubrication model is developed that describes lipid spreading and film height evolution. It is shown that lipids may play an important role in drawing the tear film up the cornea during the opening phase of the blink. Further, nonuniform distributions of lipids may lead to a rapid thinning of the tear film behind the advancing lipid front (shock). Experiments using a fluorescein dye technique and using a tearscope were undertaken in order to visualise the motion of the lipid layer and any associated shocks immediately after a blink. It is found that the lipid layer continues to spread upwards on the cornea after the opening phase of the blink, in agreement with the model. Using the experimental data, lipid particles were tracked in order to determine the surface velocity and these results are compared to the model predictions.     

MUC16 in the lacrimal apparatus

The aim of the present study was to determine the possible expression of the mucin MUC16 in the lacrimal apparatus. Expression and distribution of MUC16 in lacrimal gland, accessory lacrimal glands, and nasolacrimal ducts was monitored by RT-PCR and immunohistochemistry. MUC16 was expressed and detected in all tissues investigated. Comparable to conjunctiva and cornea it was membrane-anchored in accessory lacrimal glands whereas in lacrimal gland acinar cells and columnar cells of the nasolacrimal ducts it was stored in intracytoplasmic vesicles without membrane-association. Subepithelial serous glands of the nasolacrimal ducts revealed staining of the secretion product. Intracelluar production of MUC16 is present in lacrimal gland and epithelial cells of the nasolacrimal ducts but it is not clear whether this MUC16 is secreted. MUC16 seems to be shedded or secreted from the epithelial surface of subepithelial serous glands of the nasolacrimal ducts. Our results show that MUC16 is present in the whole lacrimal apparatus. Its distribution pattern suggests different physiological functions with regard to tear film physiology and tear outflow. Moreover, the results demonstrate the existence of so far not recognized qualitative differences in the secretion product of main lacrimal gland and accessory lacrimal glands (glands of Krause).     

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.     

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.     

In-depth analysis of the human tear proteome

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.     

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.     

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.     

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.     

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。     

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.     

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.

     

Isolation and characterization of putative trefoil peptide receptors

Mammalian trefoil factors (TFFs) constitute a group of three peptides (TFF1, TFF2 and TFF3) widely distributed in the gastrointestinal tract. Although a mucosal protection/healing effect of these peptides is well documented the mechanism of action is still unknown. A mucosal membrane extract was prepared from porcine stomach scrapings and incubated with a gel containing immobilized porcine TFF2. The affinity gel material was specifically eluted with a neutral buffer containing a high concentration of the ligand (porcine TFF2). A subsequent SDS–gel electrophoresis showed one protein with a MW of approximately 220 kDa and three proteins with MW around 140 kDa. The proteins were analyzed by trypsin digestion followed by mass spectrometric sequencing of tryptic fragments. In this way a 140-kDa β subunit of fibronectin receptor and a 224-kDa CRP-Ductin gene product were identified. The CRP-Ductin gene product (also named MUCLIN), which is expressed in the intestinal crypts, is characterized by being a membrane protein with a short cytoplasmic region, a transmembrane domain and a large extracellular region. This protein thus fulfils some of the criteria for being a TFF receptor or a TFF binding protein.     

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.     

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.