SMART MODE:

NORMAL GENOMIC

• Longman RJ et al.
• Coordinated localisation of mucins and trefoil peptides in the ulcer associated cell lineage and the gastrointestinal mucosa.
• Gut. 2000; 47: 792-800
• Display abstract

• BACKGROUND AND AIMS: Trefoil factor family (TFF) peptides and the chromosome 11p15.5 mucin glycoproteins are expressed and secreted in a site specific fashion along the length of the gastrointestinal tract. Evidence for coexpression of mucins and trefoil peptides has been suggested in numerous gastrointestinal mucosal pathologies. The ulcer associated cell lineage (UACL) occurs at sites of chronic ulceration in Crohn's disease, expresses all three trefoil peptides, and is implicated in mucosal restitution. We tested the hypothesis that individual trefoil peptides are uniquely localised with specific mucins in the UACL and normal gastrointestinal epithelia. METHODS: Expression of mucin genes in the UACL from small bowel tissue of patients with Crohn's disease was detected by in situ hybridisation, and localisation of the products by immunohistochemistry. Colocalisation of mucins and trefoil peptides was demonstrated by immunofluorescent colabelling in UACL and normal gastrointestinal epithelia. RESULTS: MUC5AC and TFF1 were colocalised in distal ductular and surface elements of the UACL and in foveolar cells of the stomach, whereas MUC6 and TFF2 were colocalised to acinar and proximal ductular structures in the UACL, in the fundus and deep antral glands of the stomach, and in Brunner's glands of the duodenum. MUC5B was found sporadically throughout the UACL and gastric body. MUC2 was absent from the UACL, Brunner's glands, and stomach. MUC2 and TFF3 were colocalised throughout the large and small bowel mucosa. CONCLUSIONS: The UACL has a unique profile of mucin gene expression. Coordinated localisation of trefoil peptides and mucins in UACL and normal gastrointestinal epithelia suggests they may assist each others' functions in protection and repair of gastrointestinal mucosa.

• Giraud AS
• X. Trefoil peptide and EGF receptor/ligand transgenic mice.
• Am J Physiol Gastrointest Liver Physiol. 2000; 278: 5016-5016
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• The use of genetically engineered mice with both gain-of-function and loss-of-function mutations has been particularly informative about the normal and pathophysiological actions of a number of regulatory peptides of the gastrointestinal tract. This review highlights some of the major findings pertinent to the epidermal growth factor (EGF) receptor and its ligands, particularly the major gut ligand transforming growth factor-alpha, as well as the trefoil peptides. Both of these peptide families have important local actions in maintaining tissue homeostasis and repair after injury, and when mechanisms governing their regulation are disrupted they may contribute to disease progression. Future applications of transgenic technology to these areas are likely to be productive in furthering our understanding of the biology of these peptides in health and disease.

• Dunbar AJ, Goddard C
• Structure-function and biological role of betacellulin.
• Int J Biochem Cell Biol. 2000; 32: 805-15
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• Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterised by a six-cysteine consensus motif that forms three intra-molecular disulfide bonds crucial for binding the ErbB receptor family. BTC was initially described, purified and cloned from a mouse insulinoma cell line. BTC is proteolytically processed from a larger membrane-anchored precursor and is a potent mitogen for a wide variety of cell types. BTC binds and activates ErbB-1 and ErbB-4 homodimers and is further characterised by its unique ability to activate all possible heterodimeric ErbB receptors. BTC is widely expressed in most tissues and various body fluids, including milk. Expression is particularly high in the pancreas where it is thought to play a role in the differentiation of pancreatic beta cells. While much is known about the ErbB receptor binding characteristics of BTC and its effect on a variety of cultured cells under different conditions, the challenge that lies ahead is to determine the role of BTC in vivo. This review will focus on the structure of BTC and the various biological effects ascribed to this member of the EGF family.

• Kinoshita K, Taupin DR, Itoh H, Podolsky DK
• Distinct pathways of cell migration and antiapoptotic response to epithelial injury: structure-function analysis of human intestinal trefoil factor.
• Mol Cell Biol. 2000; 20: 4680-90
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• The trefoil peptide intestinal trefoil factor (ITF) plays a critical role in the protection of colonic mucosa and is essential to restitution after epithelial damage. These functional properties are accomplished through coordinated promotion of cell migration and inhibition of apoptosis. ITF contains a unique three-looped trefoil motif formed by intrachain disulfide bonds among six conserved cysteine residues, which is thought to contribute to its marked protease resistance. ITF also has a seventh cysteine residue, which permits homodimer formation. A series of cysteine-to-serine substitutions and a C-terminally truncated ITF were made by PCR site-directed mutagenesis. Any alteration of the trefoil motif or truncation resulted in loss of protease resistance. However, neither an intact trefoil domain nor dimerization was required to promote cell migration. This pro-restitution activity correlated with the ability of the ITF mutants to activate mitogen-activated protein (MAP) kinase independent of phosphorylation of the epidermal growth factor (EGF) receptor. In contrast, only intact ITF retained both phosphatidylinositol 3-kinase and the EGF receptor-dependent antiapoptotic effect in HCT116 and IEC-6 cells. The inability to block apoptosis correlated with a loss of trefoil peptide-induced transactivation of the EGF receptor or Akt kinase in HT-29 cells. In addition to defining structural requirements for the functional properties of ITF, these findings demonstrate that distinct intracellular signaling pathways mediate the effects of ITF on cell migration and apoptosis.

• Sommer P, Blin N, Gott P
• Tracing the evolutionary origin of the TFF-domain, an ancient motif at mucous surfaces.
• Gene. 1999; 236: 133-6
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• Mammalian trefoil factor family (TFF)-domain peptides promote gastrointestinal protection, healing and cell migration and may act as tumour suppressors. TFF-like domains also constitute modules of composite proteins like mucin glycoproteins and zona pellucida proteins. Database searches with a modified, less stringent consensus sequence - C-x(5,6)-[ST]-x(3)-C-x(4,5)-C-C-[FYWH]-x(2, 24)-C-[FY] - revealed that ancestors of the TFF-domain arose before amphibian evolution. Eggshell proteins and a zona pellucida-like protein in teleost species, an epidermis-specific protein in a tunicate as well as an open reading frame in a nematode exhibited TFF-like motifs suggesting that they most likely originated in some multicellular organism.

• Soriano A
• [Trefoil peptides, cellular repair and progression]
• Gastroenterol Hepatol. 1999; 22: 188-90

• Brigstock DR
• The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family.
• Endocr Rev. 1999; 20: 189-206

• Bateman A, Bennett HP
• Granulins: the structure and function of an emerging family of growth factors.
• J Endocrinol. 1998; 158: 145-51
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• The granulin/epithelin motif defines a family of structurally unique proteins, of great evolutionary antiquity, which have been implicated as regulators of cell growth. Recurrent in granulin research are the surprising parallels between the granulin and EGF systems. Both are cysteinerich peptides of approximately 6 kDa that can modify cell growth. They show similar, but not identical, biological activities, although granulin/epithelin peptides do not bind EGF receptors; the three-dimensional folds of granulin and EGF are partially superimposible; and the precursors for mammalian granulin/epithelins and EGF are both organized as multiple repeats of conserved cysteine modules. Given the dissimilarity between amino acid sequences of members of the granulin/epithelin family and EGF-related peptides, the parallelism between the two systems probably represents convergent evolution towards related solutions to common biological problems. The granulin/epithelin precursor gene is expressed throughout the body, but its expression is predominantly in epithelial and haematopoietic cells. There is a great deal of versatility in the means by which cells process and handle the granulin/epithelin precursor. In some instances, the precursor is secreted intact (Zhou et al. 1993), and in others it is stored in a vesicular organelle, such as the sperm acrosome (Baba et al. 1993a). It may be processed into small 6-kDa peptides, which, in the neutrophil, can also be stored in vesicles (Bateman et al. 1990, Couto et al. 1992). The 6-kDa peptide forms, the intact precursor, and related proteins such as TGFe, regulate the growth of epithelial and mesenchymal cells. Epithelial cells express putative receptors for granulin/epithelin peptides and TGFe (Culouscou et al. 1993, Parnell et al. 1995). Thus, although much remains to be clarified, granulin/epithelin polypeptides and related proteins are emerging as widely distributed potential autocrine and paracrine growth modulating factors for epithelial and mesenchymal cells.

• Stjernquist M
• Endothelins--vasoactive peptides and growth factors.
• Cell Tissue Res. 1998; 292: 1-9
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• The endothelins (ETs) are regulatory peptides, distributed in many organ systems and with potent physiological effects. ETs represent the most powerful vasoconstrictive substances known today. They also act as growth factors and seem to be involved in fetal development. Much data support a pathophysiological role for ETs, especially in diseases of the vascular system, such as hypertension, preeclampsia, ischemic heart disease, subarachnoidal haemorrhage, and cerebral ischemia. The development of drugs affecting ET-receptors and the biosynthesis of ETs presently attract great interest for the establishment of new treatments of diseases in which ETs are involved. Hopefully, the elaboration of more specific ET-receptor ligands will fulfill some of these expectations.

• Murphy MS
• Growth factors and the gastrointestinal tract.
• Nutrition. 1998; 14: 771-4
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• This paper reviews areas of interest in gut mucosal growth factor physiology. Several epidermal growth factor (EGF)-like peptides (EGF, transforming growth factor [TGF]-alpha, heparin-binding EGF-like peptide, amphiregulin, and betacellulin) have been identified in the gut, EGF is produced by the salivary glands and is present in milk. It may act on the mucosa from the lumen as a surveillance peptide promoting mucosal repair. A stem-cell-derived "ulcer-associated cell lineage" develops adjacent to ulcers and produces EGF, which may play a role in ulcer healing. TGF-alpha is expressed by villus enterocytes and may have an important role in mucosal healing. The Trefoil peptides (pS2, spasmolytic polypeptide, intestinal trefoil factor) are protease resistant molecules secreted by mucin cells throughout the gut, with a role in mucosal healing. The TGF-beta family inhibit cell proliferation, and promote cell differentiation. TGF-beta has a gradient of expression along the crypt villus axis, with maximum production at the villus tip. It is suspected that it may prevent cell proliferation and support differentiation of villus enterocytes. Hepatocyte growth factor is a multifunctional growth factor expressed in many tissues, including the gastrointestinal tract. It has a role in organogenesis. Intestinal adaptation is highly dependent on enteral nutrition, and it is likely that growth factors are involved in adaptation. Little is known, however, about interactions between nutrients and growth factors. Milk contains a range of potentially important growth factors. Their biological significance is uncertain, and this is an area of active research.

• May FE, Westley BR
• Trefoil proteins: their role in normal and malignant cells.
• J Pathol. 1997; 183: 4-7
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• The three human trefoil proteins pS2, human intestinal trefoil factor (hITF), and human spasmolytic polypeptide (hSP) are expressed principally in the mucosa of the gastrointestinal tract. They are also expressed in a variety of other normal tissues and tumours. This review discusses the pattern of expression of trefoil proteins in cancer, current views on the biological functions of trefoil proteins, and the way in which the expression of trefoil proteins may influence the behaviour of cancer cells.

• Rossi G
• [Trefoil peptides in the protection of the gastrointestinal mucosa]
• Recenti Prog Med. 1997; 88: 201-201

• Plaut AG
• Trefoil peptides in the defense of the gastrointestinal tract.
• N Engl J Med. 1997; 336: 506-7

• Ueno N
• [Regulatory mechanisms for morphogenic factor function]
• Seikagaku. 1997; 69: 1151-65

• Thim L
• Trefoil peptides: from structure to function.
• Cell Mol Life Sci. 1997; 53: 888-903
• Display abstract

• The unique structure in which six cysteine residues in a sequence of 38 or 39 amino acid residues form three disulphide bonds in a 1-5, 2-4 and 3-6 configuration constitutes the basic elements of a trefoil domain. Today three mammalian trefoil factors (TFF1, TFF2 and TFF3) containing one or two trefoil domains are known. Trefoil factors are usually associated with the mucin layer of the gastrointestinal tract. Early studies on trefoil factors concentrated on structure elucidation and sites of expression in health and disease, whereas studies over the last 3-5 years have focused on the mechanism of action and the search for specific receptors. This review summarises our present knowledge of trefoil peptide structures, their sites of expression, and their protection and repair functions, with a focus on the mechanism by which these peptides exert their biological function.

• Playford RJ
• Trefoil peptides: what are they and what do they do?
• J R Coll Physicians Lond. 1997; 31: 37-41
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• The trefoil peptides help to protect the lining of the gastrointestinal tract. They have two beneficial roles in the gastrointestinal tract: 1. In basal circumstances, they may play a role in mucus stabilisation. 2. When an acute injury occurs, their rapid upregulation is important in stimulating the repair process, particularly that of epithelial restitution. The trefoil peptide motif consists of a unique 'three-loop' structure formed by intrachain disulphide bonds. Three members of this family have been identified in humans; spasmolytic polypeptide, intestinal trefoil factor and pS2. The trefoil peptides are expressed by mucus-producing cells throughout the normal gastrointestinal tract in a site-specific manner. In addition, the production of all three trefoil peptides is ectopically expressed in cells surrounding areas of damage in conditions such as peptic ulceration and inflammatory bowel disease.

• Nybo RE, Everton EN, Morris CF
• Mitogenic activity of keratinocyte growth factor amino-terminal truncation mutants: deletion of amino acid residues 1-15 through 1-27.
• In Vitro Cell Dev Biol Anim. 1997; 33: 606-7

• Williams GR, Wright NA
• Trefoil factor family domain peptides.
• Virchows Arch. 1997; 431: 299-304
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• Within the past 15 years a new family of peptides has been identified, known as trefoil factor family (TFF) domain peptides; they are associated with mucin-secreting epithelial cells and synthesised predominantly in the gastrointestinal tract. They share a highly conserved physical structure, and their role in mucosal defence and healing is becoming increasingly clear, more recently a tumour suppressor function has been postulated. Outside the gastrointestinal tract, members of this group of peptides have also been identified in the normal hypothalamus and pituitary, and in normal breast tissue where it is responsive to oestrogen stimulation. Evidence of peptide expression has been found in a range of urological, gynaecological, gastrointestinal, pulmonary and breast carcinomas, and in the last two it appears to carry prognostic significance. The present review aims to summarise the rapidly expanding data on the role of these peptides in epithelial inflammation, repair and neoplasia.

• Uribe JM, Barrett KE
• Nonmitogenic actions of growth factors: an integrated view of their role in intestinal physiology and pathophysiology.
• Gastroenterology. 1997; 112: 255-68

• Featherstone C
• Soothing the troubled gut with trefoil factors.
• Lancet. 1997; 349: 706-706

• Liu D et al.
• Phosphorylation of beta-catenin and epidermal growth factor receptor by intestinal trefoil factor.
• Lab Invest. 1997; 77: 557-63
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• Intestinal trefoil factor (TFF3) is a member of the trefoil family of peptides, which are constitutively expressed in the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells in vitro and to enhance epithelial restitution in vivo. In the present study, we show that the stimulatory effect of TFF3 on the migration of HT29 colonic carcinoma cells requires the perturbation of E-cadherin function, a calcium-dependent cell-cell adhesion molecule in epithelia. A rapid (< 1 minute) and specific tyrosine phosphorylation of beta-catenin and epidermal growth factor receptor was detected in cells treated with recombinant rat TFF3. No phosphorylation of E-cadherin or alpha-catenin was detected. Tyrosine phosphorylation of beta-catenin was associated with reduced membranous E-cadherin expression, perturbation of intercellular adhesion, and promotion of cell motility. These results suggest that TFF3 enhances cell migration through modulation of E-cadherin/catenin complex function. Tyrosine phosphorylation of beta-catenin and epidermal growth factor receptor seems to be involved in this process.

• Wright NA, Hoffmann W, Otto WR, Rio MC, Thim L
• Rolling in the clover: trefoil factor family (TFF)-domain peptides, cell migration and cancer.
• FEBS Lett. 1997; 408: 121-3
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• Trefoil factor family (TFF)-domain peptides 1-3 are mucin-associated molecules, largely found in epithelia of gastrointestinal tissues. Structurally similar, resistant to enzymatic degradation, they are up-regulated around areas of epithelial damage such as ulcers. Transgenic expression or exogenous peptide ameliorates or prevents gastric mucosal damage due to indomethacin and some are rapidly up-regulated after cryogenic burns. A role in promoting cell migration is strongly suggested. Knockout mice lacking TFF1 or TFF3 show significant pathology, with the former developing gastric tumours. A recent Conference Philippe Laudat agreed upon a new nomenclature for these peptides.

• Poulsom R
• Trefoil peptides.
• Baillieres Clin Gastroenterol. 1996; 10: 113-34
• Display abstract

• There is a growing body of evidence supporting the hypothesis that members of the trefoil peptide family are involved actively in maintaining the integrity of the gastrointestinal mucosa and facilitating its repair. To date, three trefoil peptides are known in man: pS2, ITF and SP. Each is a secretory peptide expressed in specific compartments throughout the gut, in patterns that appear generally to be conserved between mammalian species. Ulceration, whether due to common pathological processes or experimentally induced, results in altered local expression of trefoil peptides. In diverse chronic ulcerative conditions in man, glandular structures develop within the mucosa, derived from the UACL. These UACL glands express three trefoil peptides, EGF and lysozyme, all potentially able to contribute to the healing process. In fact local goblet and endocrine cell types may also be recruited to secrete pS2 into the local environment. In experimental ulcers, in rate stomach or intestinal resection margins, there is also accentuation of trefoil peptide expression at the margins and in the poorly differentiated mucous cells extending out presumably in attempts to restore epithelial integrity. Several trefoil peptides have been expressed as 'recombinant' proteins in bacterial, baculoviral or yeast systems, and these procedures have allowed some of the biological properties of these peptides to be determined. In vitro, rITF, hITF and hSP are motogens, able to promote migration of epithelial cells. In vivo, rITF and hSP are able to prevent much of the gastric damage effect by a single dose of indomethacin, when given systemically. There is synergy between EGF and rITF both in vitro and in vivo, which may allow the development of new peptide therapies for ulceration that will maximize repair and minimize cell proliferation.

• Liu QM, Wilkinson MC, Smith JA
• A growth factor activity in bovine milk.
• Biochem Soc Trans. 1996; 24: 342-342

• Beck S, Schmitt H, Shizuya H, Blin N, Gott P
• Cloning of contiguous genomic fragments from human chromosome 21 harbouring three trefoil peptide genes.
• Hum Genet. 1996; 98: 233-5
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• A group of small peptides with a typical cysteine-rich domain (termed trefoil motif or P-domain) is abundantly expressed at mucosal surfaces of specific normal and neoplastic tissues. Their association with the maintenance of surface integrity was suggested. The first known human trefoil peptide (pS2) was isolated from breast cancer cells (MCF7). Its oestrogen-inducible gene, and the human homologue to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localised at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene were described recently. By using suitable oligonucleotide primers and PCR and isolating large (110-250 kb) genomic recombinants cloned in the bacterial artificial chromosome (BAC) system, we present a genomic region from chromosome band 21q22.3 cloned in contiguous sequences and encoding all three members of human P-domain/trefoil peptides proving a physical linkage of all three trefoil peptide genes. Such genomic sequences will provide useful experimental material for analysis of gene regulation, for gene modification experiments and for establishing transgenic cells or animals.

• Cabral AR, Castor CW
• Connective tissue activating peptide-V and CD59: a molecule in search of a job.
• J Rheumatol. 1996; 23: 1126-9

• Poulsom R, Begos DE, Modlin IM
• Molecular aspects of restitution: functions of trefoil peptides.
• Yale J Biol Med. 1996; 69: 137-46
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• Healing of mucosal damage takes place in two phases: restitution of mucosal integrity and remodeling towards recreating the original glandular arrangements. These processes can be observed in several experimental rodent models: e.g., cryoprobe or NSAID-generated ulcers in the gastric or duodenal mucosa and following surgical resection of the small or large bowel. In some studies, it has been possible to detect changes in the expression of peptides, either in the reparative epithelium or adjacent to the damage, that may contribute to the healing processes. Trefoil peptides are expressed constitutively by epithelial cells in specific regions of the gastrointestinal tract, in association with mucins. Several studies have shown that trefoil peptide expression is enhanced at sites of damage in man and rat, and experimental evidence supports their active participation in the healing process. Recombinant trefoil peptides are able to enhance the rate of epithelial cell migration in vitro and are able to protect against indomethacin-induced damage in vivo, yet they do not depend upon TGF-beta for enhancing cell migration and do not appear to affect acid secretion. The mode of action of trefoil peptides appears to be receptor-mediated but is not simple. There is good evidence that there are interactions between members of the trefoil family and the EGF family that are beneficial for mucosal defense and repair. This raises the possibility that combining trefoil peptides with other growth factors or small molecules may be advantageous for treatment of ulceration.

• Schmitt H et al.
• A third P-domain peptide gene (TFF3), human intestinal trefoil factor, maps to 21q22.3.
• Cytogenet Cell Genet. 1996; 72: 299-302
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• Small peptides displaying a cysteine-rich module (termed P-domain or trefoil motif) form a recently increasing group of peptides abundantly expressed at mucosal surfaces of specific tissues and are associated with the maintenance of surface integrity. The estrogen-inducible pS2 gene (BCEI) and the human homolog to the porcine spasmolytic peptide (hsP) gene (SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be located at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene (TFF3) were described recently. By PCR analysis of a somatic cell hybrid panel and FISH using two large genomic recombinants (110 kb, 210 kb) cloned in the Bacterial Artificial Chromosome (BAC) system, we show that this gene coding for the new member of human P-domain/trefoil peptides also maps to chromosome region 21q22.3 suggesting a physical linkage of all three trefoil peptide genes.

• Chinery R, Coffey RJ
• Trefoil peptides: less clandestine in the intestine.
• Science. 1996; 274: 204-204

• Goodlad RA, Wright NA
• Clinical gastroenterology. Introduction.
• Baillieres Clin Gastroenterol. 1996; 10: 0-0

• Mashimo H, Podolsky DK, Fishman MC
• Structure and expression of murine intestinal trefoil factor: high evolutionary conservation and postnatal expression.
• Biochem Biophys Res Commun. 1995; 210: 31-7
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• Intestinal Trefoil Factor (ITF) is a member of a family of gastrointestinal tract peptides with region-specific expression which are enhanced at sites of injury and repair. In the present study, the murine homologue gene of ITF was molecularly cloned in order to characterize the structure and expression of this peptide in mice. Murine ITF exhibited 78, 95 and 94% nucleotide homology respectively in exons I, II and III, with overall 90% predicted amino acid identity when compared to the rat ITF. Murine ITF exhibited 70% inferred amino acid identity compared with human ITF. Northern blot analysis of various adult mouse tissues demonstrated that ITF is expressed abundantly in the intestine and colon, and minimally in stomach, but not in brain, lung, spleen, kidney, uterus, pancreas, liver, heart or thymus tissues. Expression of ITF appeared to occur as a post-natal event: antibody specific for ITF stains intensely goblet cells in the intestine and colon of three-day old and older mice, but not in the gastrointestinal tract of younger mice or embryos.

• Taupin DR, Pang KC, Green SP, Giraud AS
• The trefoil peptides spasmolytic polypeptide and intestinal trefoil factor are major secretory products of the rat gut.
• Peptides. 1995; 16: 1001-5
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• Spasmolytic polypeptide (SP) and intestinal trefoil factor (ITF) are trefoil peptides expressed by gut mucus cells. Using specific antisera we have quantified and characterized the molecular forms and distribution of these peptides in the rat gut. SP predominates in the gastric antrum as a 12 kDa form. ITF (7 kDa) is highly expressed throughout the small intestine. Both peptides are distributed in the apical secretory compartment of antral mucus cells (SP) and goblet cells (ITF), and on the lumenal surface. This study quantifies SP and ITF for the first time, and confirms them as major secretory products of the rat gut.

• Chinery R, Bates PA, De A, Freemont PS
• Characterisation of the single copy trefoil peptides intestinal trefoil factor and pS2 and their ability to form covalent dimers.
• FEBS Lett. 1995; 357: 50-4
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• A bacterial recombinant expression system was established to produce biologically active rat Intestinal Trefoil Factor (rITF). Characterisation of purified rITF shows that both monomers and dimers can be observed under reducing and non-reducing conditions, respectively. Site-directed mutagenesis studies show that Cys57 is necessary for rITF dimer formation. Samples of human gastrointestinal tissue following biopsy also demonstrated the presence of reducible human pS2 and ITF covalent dimers. Three-dimensional models for pS2 and ITF support the hypothesis that both pS2 and ITF can exist as disulphide-linked dimers in vivo and that any proposed function for these peptides must take dimer formation into account.

• Thim L
• Trefoil peptides: a new family of gastrointestinal molecules.
• Digestion. 1994; 55: 353-60

• Elia G, Chinery R, Hanby AM, Poulsom R, Wright NA
• The production and characterization of a new monoclonal antibody to the trefoil peptide human spasmolytic polypeptide.
• Histochem J. 1994; 26: 644-7
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• Human spasmolytic polypeptide (hSP) is a member of the growing family of trefoil peptides which are expressed in discrete regions of the body, most notably the gastrointestinal tract. Much of the research into the localization of the spasmolytic polypeptide has relied on hybridization in situ to detect its mRNA, due to the absence of a suitable antibody. The aim of the present study was to develop and characterize a monoclonal antibody against the human spasmolytic polypeptide, using a combination of immunohistochemistry and hybridization in situ. After immunoblotting, the antibody detected a 14 kDa protein in gastrointestinal tissue extracts from the stomach and small intestine only. Using immunohistochemistry, human spasmolytic polypeptide showed a distinctive staining pattern in the duodenum which co-localized with its mRNA. The co-localization of the immunoreactive peptide with its mRNA provides good evidence that the antibody truly recognized human spasmolytic polypeptide.

• Hauser F et al.
• hP1.B, a human P-domain peptide homologous with rat intestinal trefoil factor, is expressed also in the ulcer-associated cell lineage and the uterus.
• Proc Natl Acad Sci U S A. 1993; 90: 6961-5
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• The six-cysteine P-domain motif forms the basic repeat unit of a growing family of mucin-associated peptides. A precursor for a human secretory polypeptide has been discovered by molecular cloning and deduced to have a single P-domain, termed hP1.B. The pre-pro-peptide has 67% amino acid identity with rat intestinal trefoil factor. We find, using the techniques of RNA analysis and in situ hybridization, that this P-domain peptide is expressed in the human gastrointestinal tract, where a number of pathological conditions affect its expression, and surprisingly find it is expressed in the uterus also. In the intestine, hP1.B is expressed by goblet cells, but in Crohn disease this peptide is synthesized and secreted additionally by the ulcer-associated cell lineage that is known to secrete two other trefoil peptides, pS2 and spasmolytic polypeptide (hSP). In the stomach, hP1.B mRNA is relatively scarce but is more abundant in foci of intestinal metaplasia and near to ulceration. Mucin-rich epithelial cells in hyperplastic polyps of the colon also express this peptide. The discovery of this P-domain peptide and its expression in association with mucins support the hypothesis that P-domains with mucins may subserve related functions in the maintenance and repair of mucosal function.

• Gajhede M et al.
• Pancreatic spasmolytic polypeptide: first three-dimensional structure of a member of the mammalian trefoil family of peptides.
• Structure. 1993; 1: 253-62
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• BACKGROUND: The trefoil peptides are a rapidly growing family of peptides, mainly found in the gastrointestinal tract. There is circumstantial evidence that they stabilize the mucus layer, and may affect the rate of healing of the mucosal epithelium. RESULTS: We have determined the structure of porcine pancreatic spasmolytic polypeptide (PSP) to 2.5 A resolution. The polypeptide contains two trefoil domains. The domain structure is compact, and is composed of a central short antiparallel beta-sheet with one short helix above and one below it. This is a novel motif. The two domains are related by two-fold symmetry, and each domain contains a cleft. CONCLUSIONS: The cleft within each domain could accommodate a polysaccharide chain, and may therefore be responsible for binding mucin glycoproteins. We suggest that PSP may cross-link glycoproteins, explaining its ability to stabilize the mucus layer.

• Poulsom R, Wright NA
• Trefoil peptides: a newly recognized family of epithelial mucin-associated molecules.
• Am J Physiol. 1993; 265: 20513-20513
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• Members of the trefoil family of peptides are generally small stable secreted molecules, structurally related by the presence of one, or up to six, compact 6-cysteine motifs. Several trefoil peptides are expressed in mammalian gut and Xenopus gut and skin, often in association with mucins. Chronic ulcerative conditions of the gut, such as Crohn's disease, result in the growth of glandular structures of the ulcer-associated cell lineage (UACL) that secrete epidermal growth factor/urogastrone, transforming growth factor-alpha, and at least three trefoil peptides [pS2, human spasmolytic polypeptide (hSP), and intestinal trefoil factor (hITF/hP1.B)]. Neuroendocrine and goblet cells near the UACL are "recruited" into expressing pS2 and hSP, but the purpose of this concerted expression is unclear. A role in mucosal healing has been proposed. Biological functions of trefoil peptides have been difficult to establish. Pancreatic spasmolytic polypeptide of porcine origin inhibits gastric acid secretion and smooth muscle contraction and is a growth factor for some cultured cells, but pS2, once thought to be breast cancer specific, is not a mitogen. Recombinant trefoil peptides have allowed localization of binding sites and will allow structure-activity relationships to be studied, once the functions are clear.

• Podolsky DK et al.
• Identification of human intestinal trefoil factor. Goblet cell-specific expression of a peptide targeted for apical secretion.
• J Biol Chem. 1993; 268: 12230-12230

• Carr MD
• 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.
• Biochemistry. 1992; 31: 1998-2004
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• 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.

• Thim L
• A new family of growth factor-like peptides. 'Trefoil' disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins).
• FEBS Lett. 1989; 250: 85-90
• Display abstract

• Four peptides present in completely different biological sources have been shown to exhibit a large degree of structural similarity. The peptides include: (i) a 60 amino acid residue breast cancer associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF-7; (ii) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and pancreatic juice; and (iii) a 49 and 50 amino acid residue peptide predicted from a cDNA isolated from the skin of the frog, Xenopus laevis. These peptides are characterized by having one (pS2 and the frog peptides) or two (PSP) domains of a highly conserved 38-39 amino acid residue consensus sequence not found in any other known peptides or proteins. The domain sequences contain 6 cysteine residues in nearly the same positions and it is suggested that these 6 residues are linked by 3 disulphide bonds to form a characteristic 'trefoil' disulphide loop structure common in all four peptides. From the sources of which the peptides have been isolated and from experiments showing that PSP has a growth factor stimulatory effect on MCF-7 cells, it is further suggested that these peptides may represent members of a new family of growth factors.

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