Trefoil Motif Research Articles

Why Is There a Trefoil Motif From Karja Church in Pikk Street in Tallinn? Based on the Example of the Restoration of the In-Situ Gothic Portal in Pikk Street 7, How Medieval Is the Medieval Architecture of Tallinn Old Town?

Although Tallinn is known for its authentic medieval architecture,the closer to details we delve, to view the medieval buildings fromthe perspective of particulars, or the history of things, following themethod of its apologist Ivan Gaskell, and take one concrete artefact asa starting point and basis, the more variegated the picture becomes.The portal of the medieval building in Pikk Street 7 contains someof the more interesting restoration questions. This article here looks atthe story of the restoration of a Gothic portal of a medieval residencein Tallinn. When we stand on Pikk Street today and take a cursorylook at the portal of the house number 7, with the masonry windowson either side of it, it may seem that this medieval portal ensemble
 has survived as well as the one in Vene 17 in Tallinn. However, theportal of Pikk 7 has been chosen as the subject of this article because itcontains one of the most interesting and well-documented restorationstories of a medieval Gothic portal in Tallinn Old Town. How did ithappen that these two leave a similar impression?The article submits that the residence at Pikk 7 is indeed partiallypart of the medieval stonemasonry tradition of Tallinn, but certainlyto a lesser extent than the portal of Vene 17, which has survived in itsoriginal shape and place and has not been demolished and restored.The perspective portal of Pikk 7 is medieval to the same extent asit is from the 20th century and adds to the restoration history ofTallinn as much as to the studies of the medieval architecture ofTallinn. The Middle Ages have been the main target era and Gothicthe main target style of the restoration of the building at Pikk 7 – theend results have been aimed at the dominance of those. However, itwas decided not to restore the high gable with the blind niches thatcharacterise the medieval residences of Tallinn.To conclude, a fitting thought from Juhan Maiste, ‘Every singlething has a double meaning which tells us of the people to whomthose things once used to belong, but also of those who have cleanedand restored them, brought them, whether in their natural form oras a verbal text, back into the light.’ (Juhan Maiste, “Arvustus: Tagasi asjade juurde. Raimo Pullat. Tallinlase asjademaailmvalgustussajandil”, Akadeemia, 9 (2017), 1694.)The article is based on official restoration documentation,restoration critique in the press, and personal work memoirs ofrestorer Aarne Joonsaar.

Abstract 3161: Novel evidence for Trefoil Factor 3 as an oncogenic mediator of disease progression, and a potential predictor for neoadjuvant chemotherapy in colorectal cancer

Abstract Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. The increased incidence rates of CRC places a considerable burden on individuals and society in Asian countries. Although surgical resection remains the standard treatment for CRC patients, accumulating evidences showed advanced patients derived an additional benefit from molecular targeted agents. Therefore, there is a great need to identify novel cellular targets in CRC that can be exploited for therapeutic benefit. Trefoil factor 3 (TFF3), is a member of a family of small secretory proteins characterized by three intrachain disulfide bonds constituting the trefoil motif. TFF3 was initially found as a mediator of mucosal healing and play a critical role in epithelial restitution. Recent studies showed that TFF3 may contribute to tumorigenesis. However, its role in CRC remains unclear. Herein, the protein expression of TFF3 was measured in colorectal adenoma polyps (PLP, n=20), cancer tissues (n=20) and matched adjacent healthy tissues (n=20) by immunohistochemical (IHC) assay. We further evaluated serum and urine level of TFF3 from healthy volunteers (n=40), PLP patients (n=40) and CRC patients (n=40) by using ELISA assay. To investigate the functional or mechanistic role of TFF3 in CRC, we conducted several in vivo and in vitro approaches. The IHC showed that TFF3 was frequently overexpressed in cancer tissues (12/20, P<0.0001), while scarcely detected in PLP (2/20) or healthy tissues (3/20). In addition, serum or urine level of TFF3 was increased in CRC patients, compared to PLP patients or healthy volunteers (P=0.00032). Intriguingly, we found patients with lower serum TFF3 level are more sensitive to neoadjuvant chemotherapy than those with higher TFF3 level (with 3.4 fold increase, P<0.0001). With regard to biological function of TFF3, we found overexpression of TFF3 in CRC cell lines HCT-116 and SW480 significantly enhanced cell viability, invasion, and colony formation capacity. Subsequent mice model validated its oncogenic role in CRC, which TFF3 treatment remarkably enhances xenograft growth. In conjunction with its higher serum level in chemoresistant patients, we found overexpression of TFF3 endows HCT-116 cells with 5’-Fluorouracil resistant phenotype. Mechanistically, we observed overexpression of TFF3 is sufficient to activate Akt and Autophagy pathways in HCT-116 and SW480 cells, confirming its correlation with malignant clinical and biological phenotype. In conclusion, we identified TFF3 as a novel diagnostic marker in CRC and a promising predictor for neoadjuvant chemotherapy. Targeting TFF3 may provide an effective strategy for improving treatment of this malignancy. Note: This abstract was not presented at the meeting. Citation Format: Ying Long, Tsuyoshi Ozawa, Hanhua Li, Weijie Pan, Wenhao Weng. Novel evidence for Trefoil Factor 3 as an oncogenic mediator of disease progression, and a potential predictor for neoadjuvant chemotherapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3161.

Expression of zona pellucida B proteins in juvenile rare minnow (Gobiocypris rarus) exposed to 17α-ethinylestradiol, 4-nonylphenol and bisphenol A

Zona pellucida (ZP) containing proteins are glycoproteins in teleost chorion and are encoded by several gene subfamilies, mainly including ZPA, ZPB, ZPC and ZPX genes. In teleost species, ZP genes are expressed either in liver under regulation of estrogen or in ovary. In the present study, five ZP gene isoforms were isolated and characterized in Gobiocypris rarus. The putative amino acid sequences of these ZP gene isoforms contain the typical trefoil motif and a ZP domain. These five G. rarus ZP gene isoforms were named as grZPB.1, grZPB.2, grZPB.3, grZPB.4 and grZPB.5. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis indicated that all these ZP mRNA isoforms were exclusively expressed in ovary. G. rarus juveniles at the age of 21days postfertilization were exposed to 17α-ethinylestradiol (EE2; 0.01, 0.1 and 1nM), 4-nonylphenol (4-NP; 10, 100 and 1000 nM) or bisphenol A (BPA; 0.1, 1 and 10nM) for 3days. mRNA expressions of ZPB isoforms following the exposure to xenoestrogen were detected by RT-qPCR. Data were analyzed by the 2-△△Cq method. The results indicate that induction by 0.1–1nM EE2 on mRNA expression of the grZPB isoforms is weaker than for vitellogenin. 4-NP exposures at three concentrations had differential effects on the grZPBs. BPA at three concentrations weakly induced mRNA expression of the grZPB isoforms.

New member of the trefoil factor family of proteins is an α-macroglobulin protease inhibitor

The amino acid sequence of the monomeric α-macroglobulin (αM) from the American bullfrog, Rana catesbiana, was determined. The mature protein consisted of 1469 amino acid residues and shared sequence identity with other members of the αM family of protein. The central portion of the frog monomeric αM contained Cys residues positioned analogously to the Cys residues in human α 2-macroglobulin (α 2M), known to be involved in disulfide bridges. Additionally, the frog monomeric αM contained six Cys residues in a ∼60 residue COOH-terminal extension not present in previously characterized αMs. The spacing of the Cys residues and the overall sequence identity of this COOH-terminal extension were consistent with a trefoil motif. This is the first time a member of the trefoil factor family has been identified in the circulatory system. The “bait region” was located between Arg 675–Lys 685 and contained mainly basic amino acid residues. The COOH-terminal receptor-binding domain was not exposed prior to proteolysis of this highly susceptible region. The proximity of the receptor-binding and trefoil domains implied that the trefoil domain is similarly concealed before bait region cleavage.

Trefoil peptides in airway epithelium: an important addition to the plethora of peptides.

An increasing number of regulatory peptides have been demonstrated to be expressed in airway epithelium; these modulate various aspects of proliferation, motility and secretory function. These recognized peptides include epidermal growth factor (EGF), transforming growth factor beta (TGF), the insulin-like growth factor family, the fibroblast growth factor family, a few other seemingly unrelated regulatory peptides, such as hepatocyte growth factor, plateletderived growth factor, and various interleukins, interferons and tumor necrosis factor-related proteins. Many of these peptides have roles in other epithelial structures such as intestine and breast, and insights gained in these tissues have been useful in understanding airway function. Over the past twenty years, one family of peptides has been identified as an important regulator of gastric and intestinal epithelial repair and motility. Known as the trefoil factor family (TFF), their abundant expression in both normal and neoplastic tissues has stimulated substantial interest in understanding their regulatory roles in cell motility and mucous secretion. The trefoil family shares a variable number of conserved motifs, usually 39 to 43 residues each, based on a compact three-dimensional trefoil structure of six cysteine residues of almost invariant spacing held together by three pairs of disulfide bonds (1, 2). This structure gives the peptide a characteristic three-loop shape (Figure 1) and provides for exceptional resistance to acid digestion and proteolytic degradation. Other conserved residues have been shown to form a hydrophobic patch on the surface of the trefoil domain which may provide a binding cleft for either a sugar group or an aromatic amino acid side-chain during intermolecular interactions. Three trefoil peptides are currently recognized in humans: TFF1 (also known as pS2) and TFF3 (intestinal trefoil factor), which each have a single trefoil motif and both of which homodimerize, and TFF2 (human spasmolytic polypeptide, hSP), which has two motifs and does not require dimerization for its activity (Williams and Wright, 1997). Each map to the same cluster on chromosome 21 (3, 4). TFF2 is glycosylated via an N-linkage, and TFF2 polymorphisms are known to exist (5). The trefoil motifs in TFF2 form a tight structure compared to the loose structure formed by the dimerization of TFF1; this may lead to differences in binding and function (6). The trefoil peptides localize predominately to the gastrointestinal tract (7). Each of the peptides are found in the intestinal tract within the ductal lumenal cells of Brunner’s glands and in goblet cells near the surface of crypts. Both TFF1 and TFF2 are also found within the stomach in mucous neck cells of the antrum and fundus and in the epithelium of the pancreas and gall bladder. Peptides are packaged by the Golgi apparatus in mucin-producing cells and secreted with mucous into the protective layer above the epithelium. TFF3 also co-localizes with oxytocin-producing cells in the hypothalamus (8) and is found in epithelial cells of the female genitourinary tract (7). TFF1 is expressed in rat hippocampus, cortex, and cerebellum (9), in cultured mouse astrocytes (10) and normal breast ductal epithelium (11). TFF2 has not been reported to be expressed in normal tissues outside the gastrointestinal tract. TFF1 was originally isolated from human breast carcinoma cell lines (12, 13). Pathologic expression of this trefoil peptide since has been demonstrated in a variety of adenocarcinomas, especially those with a mucinous histology, in breast, lung, endometrium, ovary, pancreas, stomach and intestine (summarized in 14). Expression in breast carcinoma is associated with estrogen receptor presence and a favorable clinical outcome (15–17). Expression in lung adenocarcinoma is common, though expression in nonmalignant adenomas, squamous cell carcinoma, and small cell carcinoma is not seen (18). Expression in lung adenocarcinoma, in contrast to breast adenocarcinoma, has been associated with a poorer prognosis (19). The abundance of trefoil peptides in gastrointestinal malignancies is exactly opposite that seen with breast and lung malignancies. TFF1 abundance is decreased in gastric carcinomas and is lost completely in about 50% of these neoplasms (20). A similar decrease in trefoil peptide abundance has been demonstrated in colonic tumors, though complete disappearance is rare (21). The reasons for the wide discrepancies in abundance and prognosis await exploration. The physiological functions of the trefoil peptides are several, and can be divided into two broad categories: mucosal surface protection and repair after injury. Trefoil peptides interact with mucins (22) and regulate mucous viscosity (23), and via these actions may enhance the protective capabilities of the mucosal defense barrier. Both glycosylated and non-glycosylated TFF2 is found in gastric secretions with a diurnal variation, highest at night, suggesting that the maximum cytoprotective effects of trefoil peptides ( Received in original form August 22, 2001 )

Molecular Basis of Aberrant Apical Protein Transport in an Intestinal Enzyme Disorder

The impaired sorting profile to the apical membrane of human intestinal sucrase-isomaltase is the underlying cause in the pathogenesis of a novel phenotype of intestinal congenital sucrase-isomaltase deficiency. Molecular characterization of this novel phenotype reveals a point mutation in the coding region of the sucrase-isomaltase (SI) gene that results in an amino acid substitution of a glutamine by arginine at residue 117 of the isomaltase subunit. This substitution is located in a domain revealing features of a trefoil motif or a P-domain in immediate vicinity of the heavily O-glycosylated stalk domain. Expression of the mutant SI phenotype in epithelial Madin-Darby canine kidney cells reveals a randomly targeted SI protein to the apical and basolateral membranes confirming an exclusive role of the Q117R mutation in generating this phenotype. Unlike wild type SI, the mutant protein is completely extractable with Triton X-100 despite the presence of O-glycans that serve in the wild type protein as an apical sorting signal and are required for the association of SI with detergent-insoluble lipid microdomains. Obviously the O-glycans are not adequately recognized in the context of the mutant SI, most likely due to altered folding of the P-domain that ultimately affects the access of the O-glycans to a putative sorting element.

Distinct pathways of cell migration and antiapoptotic response to epithelial injury: structure-function analysis of human intestinal trefoil factor.

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.

A novel 25 bp tandem repeat within the human trefoil peptide gene TFF2 in 21q22.3: polymorphism and mammalian evolution.

Trefoil peptides belong to a family of small secretory proteins characterised by three intrachain disulfide bonds forming the trefoil motif (TFF-domain). They serve to maintain or repair the epithelial mucosa, and promote cell migration. They are predominantly found in gastrointestinal tissues, and are upregulated around areas of epithelial damage, and in meta- and neoplasia. The corresponding three genes are clustered in human 21q22.3. TFF2 is the only human one encoding two TFF-domains on separate exons. In between (intron 2), a novel 25 bp sequence is located that is tandemly repeated approximately 48 times, but is unique in the human genome. A diallelic polymorphism with a second allele comprising approximately 53 repeat units is present among individuals. Both alleles were cloned on BAC recombinants. In Caucasians (n = 78) the allele frequencies found are 0.85 and 0.15, respectively, representing a frequency of heterozygosity of 26%. Ape and monkey species exhibit homologous repeats of shorter total array length, whereas none is found in other mammals.

High-resolution solution structure of human pNR-2/ps2: A single trefoil motif protein

pNR-2/pS2 is a 60 residue extracellular protein, which was originally discovered in human breast cancer cells, and subsequently found in other tumours and normal gastric epithelial cells. We have determined the three-dimensional solution structure of a C58S mutant of human pNR-2/pS2 using 639 distance and 137 torsion angle constraints obtained from analysis of multidimensional NMR spectra. A series of simulated annealing calculations resulted in the unambiguous determination of the protein’s disulphide bonding pattern and produced a family of 19 structures consistent with the constraints. The peptide contains a single “trefoil” sequence motif, a region of about 40 residues with a characteristic sequence pattern, which has been found, either singly or as a repeat, in about a dozen extracellular proteins. The trefoil domain contains three disulphide bonds, whose 1-5, 2-4 and 3-6 cysteine pairings form the structure into three closely packed loops with only a small amount of secondary structure, which consists of a short α-helix packed against a two-stranded antiparallel β-sheet. The structure of the domain is very similar to those of the two trefoil domains that occur in porcine spasmolytic polypeptide (PSP), the only member of the trefoil family whose three-dimensional structure has been previously determined. Outside the trefoil domain, which forms the compact “head” of the molecule, the N and C-terminal strands are closely associated, forming an extended “tail”, which has some β-sheet character for part of its length and which becomes more disordered towards the termini as indicated by 15N{ 1H} NOEs. We have considered the structural implications of the possible formation of a native C58-C58 disulphide-bonded homodimer. Comparison of the surface features of pNR-2/pS2 and PSP, and consideration of the sequences of the other human trefoil domains in the light of these structures, illuminates the possible role of specific residues in ligand/receptor binding.

The trefoil peptide family.

The unique three-loop structure of the trefoil motif, formed by intrachain disulfide bonds in a 1-5, 2-4, 3-6 configuration between six conserved cysteine residues, is the defining feature of a recently recognized family of peptides. Expression of trefoil peptides is closely related to that of mucin glycoproteins in diverse biological sources. Three distinct members of the family (pS2, intestinal trefoil factor, and spasmolytic polypeptide) are produced in the mammalian gastrointestinal tract by mucus-secreting cells and targeted primarily for luminal secretion. The compact structure of the trefoil motif may be responsible for marked resistance of trefoil peptides to proteolytic digestion, enabling them to function in the harsh environment of the gastrointestinal lumen. Trefoil peptides are ectopically expressed adjacent to areas of inflammation within the gastrointestinal tract and may play an important role in both maintaining the barrier function of mucosal surfaces and facilitating healing after injury.

Cloning of contiguous genomic fragments from human chromosome 21 harbouring three trefoil peptide genes.

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.

Deciphering the Indus Script

Part I. Introduction: 1. The Indus Civilization and its historical context Part II. The Indus Script: 2. Early writing systems 3. Deciphering an unknown script 4. Approaches to the Indus script 5. Internal evidence for the type of script used in the Indus Valley 6. Internal evidence on the structure of the Indus language 7. External clues to the Indus script Part III. The Linguistic Context: 8. In search of the Indus language 9. Dravidian languages and the Harappan culture Part IV. Interpretations of Indus Pictograms: 10. The 'fish' signs of the Indus script 11. The astronomical and astrological background 12. The trefoil motif: further evidence for astral religion 13. Evidence for Harappan worship of the god Muruku 14. Evidence for Harappan worship of the Goddess 15. Epilogue Appendix Bibliographical notes References Index.

Human trefoil peptides: genomic structure in 21q22.3 and coordinated expression.

Trefoil peptides are small secretory proteins characterized by three intrachain disulfide bonds forming the trefoil motif or P-domain. They are abundantly expressed on mucosal surfaces, especially of the gastrointestinal tract. In pathological conditions such as ulcers, metaplasia and neoplasia, their expression is upregulated. Three human trefoil peptides have been described: the estrogen-inducible pS2 protein, the spasmolytic protein and the intestinal trefoil factor. Recently, their role in the maintenance of surface integrity and ulcer healing was discussed. We already mapped the corresponding three genes (BCEI), SML1, TFF3) to the same genomic region (21q22.3). In this paper, we show that the three genes are clustered in a tandemly orientated fashion within 50 kb on a bacterial artificial chromosome (BAC) recombinant. This cluster is located adjacent to D21S19 and the locus order is cen-D21S212-TFF3-SML1-BCEI-D21S19-tel, whereas transcription of all three genes is directed towards the centromere. The gene structure of SML1 exhibits four exons, two of which encode the two separate trefoil motifs. TFF3 and BCEI, both containing one trefoil motif, are composed of three exons each, suggesting gene duplication and exon-shuffling events during evolution. The 5'-flanking region of SML1 was compared to the corresponding region of other trefoil genes. Two motifs with identical sequence and positions are shared between SML1 and BCEI, thus presenting possible targets for stomach-specific gene regulation. Two other motifs are shared within all known human and rat trefoil genes, suggesting a coordinated regulation and/or a common locus-controlling region. Using RT-PCR, a change in the pattern of trefoil gene expression is detected in tissue samples from normal gastric mucosa, hyperplastic polyps, gastric cancer, and gastric cancer cell lines, respectively.

A third P-domain peptide gene (TFF3), human intestinal trefoil factor, maps to 21 q22.3

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.

Solution structure of a trefoil-motif-containing cell growth factor, porcine spasmolytic protein.

The porcine spasmolytic protein (pSP) is a 106-residue cell growth factor that typifies a family of eukaryotic proteins that contain at least one copy of an approximately 40-amino acid protein domain known as the trefoil motif. In fact, pSP contains two highly homologous trefoil domains. We have determined the complete three-dimensional solution structure of pSP by using a combination of two- and three-dimensional 1H NMR spectroscopy and distance geometry calculations. pSP is a relatively elongated molecule, consisting of two compact globular domains joined via a small interface. The protein's two trefoil domains adopt the same tertiary structure and contain a core C-terminal two-stranded antiparallel beta-sheet, preceded by a 6-residue helix that packs against the N-terminal beta-strand. The remainder of the protein backbone is taken up by two short loops that lie on either side of the beta-hairpin and are linked by an extended region that wraps around the C-terminal beta-strand. The topology of the protein backbone observed for the trefoil domains in pSP represents an unusual polypeptide fold. A striking feature of both trefoil domains is a surface patch formed from five conserved residues that have no obvious structural role. The two patches are located at the far ends of the protein molecule, and we propose that these residues form at least part of the receptor binding site, or sites, on pSP.

Crystal structure of a disulfide-linked "trefoil" motif found in a large family of putative growth factors.

Porcine pancreatic spasmolytic polypeptide (PSP) belongs to a large family of homologous growth factor-like polypeptides characterized by a disulfide-linked "trefoil motif," duplicated and conserved in various family members. PSP contains two trefoil motifs, has several pharmacological actions on the gut, and has growth factor properties on epithelial cells in vitro. The human PSP analogue, human spasmolytic polypeptide, appears to be involved in many regenerative situations and, especially, in healing gastrointestinal ulcers. One member of the trefoil family, pS2, is secreted in approximately 50% of estrogen-dependent human breast carcinomas, which has led to its use as a tumor prognostic marker. Both pS2 and human spasmolytic polypeptide are also widely expressed in chronic gastrointestinal ulcerative conditions such as Crohn disease. Here we report the three-dimensional structure at 2.6-A resolution of a trefoil-containing protein, namely PSP, purified from porcine pancreas. The structure shows two homologous domains that share a supersecondary structure and disulfide bond pattern. The two domains pack asymmetrically giving rise to a number of protruding loops, exposed clefts, and an unusual electrostatic surface potential. Knowledge of the structure of PSP should allow the design of mutants to investigate further the function of PSP and other trefoil-containing peptides.

The P-domain or trefoil motif: a role in renewal and pathology of mucous epithelia?

By analogy with epidermal growth factor and EGF-like repeats, the P-domain, or trefoil motif, is a characteristic shuffled module containing six invariant cysteine residues that forms the basic unit for a family of mucin-associated peptides. These P-domain peptides are potential modulators of cell growth and they are also expressed under certain pathological conditions. Furthermore, P-domains have been found as components of extracellular mosaic proteins including certain mucins, where they are thought to play a role either in protein-protein or in lectin-like interactions.

Expression and purification of a trefoil peptide motif in a beta-galactosidase fusion protein and its use to search for trefoil-binding sites.

The cysteine-rich trefoil motif of rat intestinal trefoil factor (rITF) was cloned and expressed in Escherichia coli. A 270-bp cDNA fragment including the signal sequence and the trefoil motif was cloned into the expression vector pAX5+ to direct the expression of a beta-galactosidase collagen-hinged fusion protein in E. coli. Cultures harbouring the recombinant plasmid produced a soluble novel protein with a molecular mass of 134.5 kDa, as predicted for the trefoil-motif-containing fusion protein. Purification of the rITF moiety was achieved by p-aminophenyl-thio-beta-D-galactoside(APTG)-affinity chromatography, collagenase digestion of the hybrid molecule, and removal of the beta-galactosidase-hinge molecule by a further APTG-affinity step. It was demonstrated that intrachain disulphide-bond formation in rITF occurred during the procedure, so no refolding steps were required. Analysis by immunoblotting revealed that the fusion protein and the cleaved trefoil-motif-containing protein were recognised by an antibody raised against the chemically synthesised peptide. The trefoil motif present in the fusion protein was used to localise putative trefoil-binding sites in sections of frozen rat tissue. Binding was demonstrated using the beta-galactosidase portion of the fusion protein as a reporter moiety, either directly with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside, or indirectly using a monoclonal antibody to beta-galactosidase and indirect immunohistochemistry. Binding sites were localised to the foveolar and surface epithelium of rat stomach, the collecting ducts of the kidney and within colonic crypts. The presence of a trefoil motif was necessary for binding. The use of beta-galactosidase fusion proteins for histochemical localisation of peptide-binding sites should prove more generally useful.

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.