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. 1997 Oct 1;327(Pt 1):117–123. doi: 10.1042/bj3270117

Homodimerization and hetero-oligomerization of the single-domain trefoil protein pNR-2/pS2 through cysteine 58.

M P Chadwick 1, B R Westley 1, F E May 1
PMCID: PMC1218770  PMID: 9355742

Abstract

The single-domain human trefoil proteins [pNR-2/pS2 and human intestinal trefoil factor (hITF)] have seven cysteine residues, of which six are involved in maintaining the structure of the trefoil domain. The seventh does not form part of the trefoil domain and is located three residues from the C-terminus. The ability of the pNR-2/pS2 single trefoil domain protein to dimerize was examined by using recombinant protein with either a cysteine or a serine residue at this position by equilibrium ultracentrifugation, laser-assisted desorption MS, gel filtration and PAGE. pNR-2/pS2 Cys58 formed dimers, whereas pNR-2/pS2 Ser58 did not. Experiments in which the dimer was treated with thiol agents demonstrated that the dimer was linked via a disulphide bond and that the intermolecular disulphide bond was more susceptible to reduction than the intramolecular disulphide bonds. To examine whether dimeric pNR-2/pS2 was secreted by oestrogen-responsive breast cancer cells, which are known to express pNR-2/pS2 mRNA, conditioned medium was separated on non-denaturing polyacrylamide gels, transferred to PVDF membrane and reacted with antiserum against pNR-2/pS2. Monomeric and dimeric pNR-2/pS2 were detected but the majority of the protein reactivity was associated with a larger protein. Treatment of this protein with thiol agents suggested that it is an oligomer containing pNR-2/pS2 linked to another protein by a disulphide bond. These studies suggest that the biological action of pNR-2/pS2 single-domain trefoil protein might involve the formation of homodimers or oligomers with other proteins.

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Selected References

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  1. Berry M., Nunez A. M., Chambon P. Estrogen-responsive element of the human pS2 gene is an imperfectly palindromic sequence. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1218–1222. doi: 10.1073/pnas.86.4.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carr M. D., Bauer C. J., Gradwell M. J., Feeney J. Solution structure of a trefoil-motif-containing cell growth factor, porcine spasmolytic protein. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2206–2210. doi: 10.1073/pnas.91.6.2206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chadwick M. P., May F. E., Westley B. R. Production and comparison of mature single-domain 'trefoil' peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58. Biochem J. 1995 Jun 15;308(Pt 3):1001–1007. doi: 10.1042/bj3081001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chinery R., Bates P. A., De A., Freemont P. S. Characterisation of the single copy trefoil peptides intestinal trefoil factor and pS2 and their ability to form covalent dimers. FEBS Lett. 1995 Jan 2;357(1):50–54. doi: 10.1016/0014-5793(94)01297-e. [DOI] [PubMed] [Google Scholar]
  5. Chinery R., Cox H. M. Immunoprecipitation and characterization of a binding protein specific for the peptide, intestinal trefoil factor. Peptides. 1995;16(4):749–755. doi: 10.1016/0196-9781(95)00045-l. [DOI] [PubMed] [Google Scholar]
  6. Chinery R., Playford R. J. Combined intestinal trefoil factor and epidermal growth factor is prophylactic against indomethacin-induced gastric damage in the rat. Clin Sci (Lond) 1995 Apr;88(4):401–403. doi: 10.1042/cs0880401. [DOI] [PubMed] [Google Scholar]
  7. De A., Brown D. G., Gorman M. A., Carr M., Sanderson M. R., Freemont P. S. Crystal structure of a disulfide-linked "trefoil" motif found in a large family of putative growth factors. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):1084–1088. doi: 10.1073/pnas.91.3.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dignass A., Lynch-Devaney K., Kindon H., Thim L., Podolsky D. K. Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway. J Clin Invest. 1994 Jul;94(1):376–383. doi: 10.1172/JCI117332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Foekens J. A., Portengen H., Look M. P., van Putten W. L., Thirion B., Bontenbal M., Klijn J. G. Relationship of PS2 with response to tamoxifen therapy in patients with recurrent breast cancer. Br J Cancer. 1994 Dec;70(6):1217–1223. doi: 10.1038/bjc.1994.476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Foekens J. A., van Putten W. L., Portengen H., de Koning H. Y., Thirion B., Alexieva-Figusch J., Klijn J. G. Prognostic value of PS2 and cathepsin D in 710 human primary breast tumors: multivariate analysis. J Clin Oncol. 1993 May;11(5):899–908. doi: 10.1200/JCO.1993.11.5.899. [DOI] [PubMed] [Google Scholar]
  11. Frandsen E. K., Jørgensen K. H., Thim L. Receptor binding of pancreatic spasmolytic polypeptide (PSP) in rat intestinal mucosal cell membranes inhibits the adenylate cyclase activity. Regul Pept. 1986 Dec 30;16(3-4):291–297. doi: 10.1016/0167-0115(86)90028-5. [DOI] [PubMed] [Google Scholar]
  12. Gajhede M., Petersen T. N., Henriksen A., Petersen J. F., Dauter Z., Wilson K. S., Thim L. Pancreatic spasmolytic polypeptide: first three-dimensional structure of a member of the mammalian trefoil family of peptides. Structure. 1993 Dec 15;1(4):253–262. doi: 10.1016/0969-2126(93)90014-8. [DOI] [PubMed] [Google Scholar]
  13. Hanby A. M., Poulsom R., Singh S., Elia G., Jeffery R. E., Wright N. A. Spasmolytic polypeptide is a major antral peptide: distribution of the trefoil peptides human spasmolytic polypeptide and pS2 in the stomach. Gastroenterology. 1993 Oct;105(4):1110–1116. doi: 10.1016/0016-5085(93)90956-d. [DOI] [PubMed] [Google Scholar]
  14. Hauser F., Hoffmann W. P-domains as shuffled cysteine-rich modules in integumentary mucin C.1 (FIM-C.1) from Xenopus laevis. Polydispersity and genetic polymorphism. J Biol Chem. 1992 Dec 5;267(34):24620–24624. [PubMed] [Google Scholar]
  15. Henry J. A., Bennett M. K., Piggott N. H., Levett D. L., May F. E., Westley B. R. Expression of the pNR-2/pS2 protein in diverse human epithelial tumours. Br J Cancer. 1991 Oct;64(4):677–682. doi: 10.1038/bjc.1991.380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henry J. A., Piggott N. H., Mallick U. K., Nicholson S., Farndon J. R., Westley B. R., May F. E. pNR-2/pS2 immunohistochemical staining in breast cancer: correlation with prognostic factors and endocrine response. Br J Cancer. 1991 Apr;63(4):615–622. doi: 10.1038/bjc.1991.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lefebvre O., Chenard M. P., Masson R., Linares J., Dierich A., LeMeur M., Wendling C., Tomasetto C., Chambon P., Rio M. C. Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science. 1996 Oct 11;274(5285):259–262. doi: 10.1126/science.274.5285.259. [DOI] [PubMed] [Google Scholar]
  18. Mashimo H., Wu D. C., Podolsky D. K., Fishman M. C. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science. 1996 Oct 11;274(5285):262–265. doi: 10.1126/science.274.5285.262. [DOI] [PubMed] [Google Scholar]
  19. Masiakowski P., Breathnach R., Bloch J., Gannon F., Krust A., Chambon P. Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human breast cancer cell line. Nucleic Acids Res. 1982 Dec 20;10(24):7895–7903. doi: 10.1093/nar/10.24.7895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. May F. E., Westley B. R. Cloning of estrogen-regulated messenger RNA sequences from human breast cancer cells. Cancer Res. 1986 Dec;46(12 Pt 1):6034–6040. [PubMed] [Google Scholar]
  21. May F. E., Westley B. R. Identification and characterization of estrogen-regulated RNAs in human breast cancer cells. J Biol Chem. 1988 Sep 15;263(26):12901–12908. [PubMed] [Google Scholar]
  22. Piggott N. H., Henry J. A., May F. E., Westley B. R. Antipeptide antibodies against the pNR-2 oestrogen-regulated protein of human breast cancer cells and detection of pNR-2 expression in normal tissues by immunohistochemistry. J Pathol. 1991 Feb;163(2):95–104. doi: 10.1002/path.1711630204. [DOI] [PubMed] [Google Scholar]
  23. Playford R. J., Marchbank T., Chinery R., Evison R., Pignatelli M., Boulton R. A., Thim L., Hanby A. M. Human spasmolytic polypeptide is a cytoprotective agent that stimulates cell migration. Gastroenterology. 1995 Jan;108(1):108–116. doi: 10.1016/0016-5085(95)90014-4. [DOI] [PubMed] [Google Scholar]
  24. Playford R. J., Marchbank T., Goodlad R. A., Chinery R. A., Poulsom R., Hanby A. M. Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):2137–2142. doi: 10.1073/pnas.93.5.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Polshakov V. I., Frenkiel T. A., Westley B., Chadwick M., May F., Carr M. D., Feeney J. NMR-based structural studies of the pNR-2/pS2 single domain trefoil peptide. Similarities to porcine spasmolytic peptide and evidence for a monomeric structure. Eur J Biochem. 1995 Nov 1;233(3):847–855. doi: 10.1111/j.1432-1033.1995.847_3.x. [DOI] [PubMed] [Google Scholar]
  26. Polshakov V. I., Williams M. A., Gargaro A. R., Frenkiel T. A., Westley B. R., Chadwick M. P., May F. E., Feeney J. High-resolution solution structure of human pNR-2/pS2: a single trefoil motif protein. J Mol Biol. 1997 Mar 28;267(2):418–432. doi: 10.1006/jmbi.1997.0896. [DOI] [PubMed] [Google Scholar]
  27. Rio M. C., Bellocq J. P., Daniel J. Y., Tomasetto C., Lathe R., Chenard M. P., Batzenschlager A., Chambon P. Breast cancer-associated pS2 protein: synthesis and secretion by normal stomach mucosa. Science. 1988 Aug 5;241(4866):705–708. doi: 10.1126/science.3041593. [DOI] [PubMed] [Google Scholar]
  28. Rio M. C., Bellocq J. P., Gairard B., Rasmussen U. B., Krust A., Koehl C., Calderoli H., Schiff V., Renaud R., Chambon P. Specific expression of the pS2 gene in subclasses of breast cancers in comparison with expression of the estrogen and progesterone receptors and the oncogene ERBB2. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9243–9247. doi: 10.1073/pnas.84.24.9243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schwartz L. H., Koerner F. C., Edgerton S. M., Sawicka J. M., Rio M. C., Bellocq J. P., Chambon P., Thor A. D. pS2 expression and response to hormonal therapy in patients with advanced breast cancer. Cancer Res. 1991 Jan 15;51(2):624–628. [PubMed] [Google Scholar]
  30. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  31. Thim L. Trefoil peptides: a new family of gastrointestinal molecules. Digestion. 1994;55(6):353–360. doi: 10.1159/000201165. [DOI] [PubMed] [Google Scholar]
  32. Tomasetto C., Wolf C., Rio M. C., Mehtali M., LeMeur M., Gerlinger P., Chambon P., Lathe R. Breast cancer protein PS2 synthesis in mammary gland of transgenic mice and secretion into milk. Mol Endocrinol. 1989 Oct;3(10):1579–1584. doi: 10.1210/mend-3-10-1579. [DOI] [PubMed] [Google Scholar]
  33. Westley B., Knowland J. An estrogen receptor from Xenopus laevis liver possibly connected with vitellogenin synthesis. Cell. 1978 Oct;15(2):367–374. doi: 10.1016/0092-8674(78)90005-3. [DOI] [PubMed] [Google Scholar]
  34. Williams R., Stamp G. W., Gilbert C., Pignatelli M., Lalani E. N. pS2 transfection of murine adenocarcinoma cell line 410.4 enhances dispersed growth pattern in a 3-D collagen gel. J Cell Sci. 1996 Jan;109(Pt 1):63–71. doi: 10.1242/jcs.109.1.63. [DOI] [PubMed] [Google Scholar]
  35. Wright N. A., Poulsom R., Stamp G. W., Hall P. A., Jeffery R. E., Longcroft J. M., Rio M. C., Tomasetto C., Chambon P. Epidermal growth factor (EGF/URO) induces expression of regulatory peptides in damaged human gastrointestinal tissues. J Pathol. 1990 Dec;162(4):279–284. doi: 10.1002/path.1711620402. [DOI] [PubMed] [Google Scholar]

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