Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon (original) (raw)

Abstract

Purification and amino acid sequence analysis of a proteolytic fragment of fibronectin (FN) from transformed human cells demonstrated that a high percentage of these FN molecules contains an extra amino acid sequence which is present only in a very low percentage of FN molecules from normal fibroblasts and is undetectable in plasma FN. This new amino acid sequence introduces into the FN molecule a site very sensitive to a number of proteolytic enzymes. By analyzing the cellular mRNA and genomic clones, we have demonstrated that this sequence derives from a differential splicing pattern of the FN mRNA precursors, which leads in transformed cells to a high-level expression of an extra type III homology repeat (ED-B) coded for by a previously unobserved exon. Here we also report the complete sequence of this new exon. These results demonstrate that in malignant cells the mechanisms regulating the splicing of FN mRNA precursors are altered.

2337

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alitalo K., Vaheri A. Pericellular matrix in malignant transformation. Adv Cancer Res. 1982;37:111–158. doi: 10.1016/s0065-230x(08)60883-0. [DOI] [PubMed] [Google Scholar]
  2. Balian G., Click E. M., Crouch E., Davidson J. M., Bornstein P. Isolation of a collagen-binding fragment from fibronectin and cold-insoluble globulin. J Biol Chem. 1979 Mar 10;254(5):1429–1432. [PubMed] [Google Scholar]
  3. Borsi L., Allemanni G., Castellani P., Rosellini C., Di Vinci A., Zardi L. Structural differences in the cell binding region of human fibronectin molecules isolated from cultured normal and tumor-derived human cells. FEBS Lett. 1985 Nov 11;192(1):71–74. doi: 10.1016/0014-5793(85)80045-4. [DOI] [PubMed] [Google Scholar]
  4. Borsi L., Carnemolla B., Castellani P., Rosellini C., Vecchio D., Allemanni G., Chang S. E., Taylor-Papadimitriou J., Pande H., Zardi L. Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells. J Cell Biol. 1987 Mar;104(3):595–600. doi: 10.1083/jcb.104.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borsi L., Castellani P., Balza E., Siri A., Pellecchia C., De Scalzi F., Zardi L. Large-scale procedure for the purification of fibronectin domains. Anal Biochem. 1986 Jun;155(2):335–345. doi: 10.1016/0003-2697(86)90443-4. [DOI] [PubMed] [Google Scholar]
  6. Castellani P., Siri A., Rosellini C., Infusini E., Borsi L., Zardi L. Transformed human cells release different fibronectin variants than do normal cells. J Cell Biol. 1986 Nov;103(5):1671–1677. doi: 10.1083/jcb.103.5.1671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  8. Colombi M., Barlati S., Kornblihtt A., Baralle F. E., Vaheri A. A family of fibronectin mRNAs in human normal and transformed cells. Biochim Biophys Acta. 1986 Dec 18;868(4):207–214. doi: 10.1016/0167-4781(86)90056-4. [DOI] [PubMed] [Google Scholar]
  9. Humphries M. J., Akiyama S. K., Komoriya A., Olden K., Yamada K. M. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J Cell Biol. 1986 Dec;103(6 Pt 2):2637–2647. doi: 10.1083/jcb.103.6.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hynes R. Molecular biology of fibronectin. Annu Rev Cell Biol. 1985;1:67–90. doi: 10.1146/annurev.cb.01.110185.000435. [DOI] [PubMed] [Google Scholar]
  11. Hörmann H. Fibronectin--mediator between cells and connective tissue. Klin Wochenschr. 1982 Oct 15;60(20):1265–1277. doi: 10.1007/BF01727483. [DOI] [PubMed] [Google Scholar]
  12. Kornblihtt A. R., Umezawa K., Vibe-Pedersen K., Baralle F. E. Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 1985 Jul;4(7):1755–1759. doi: 10.1002/j.1460-2075.1985.tb03847.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kornblihtt A. R., Vibe-Pedersen K., Baralle F. E. Human fibronectin: cell specific alternative mRNA splicing generates polypeptide chains differing in the number of internal repeats. Nucleic Acids Res. 1984 Jul 25;12(14):5853–5868. doi: 10.1093/nar/12.14.5853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kornblihtt A. R., Vibe-Pedersen K., Baralle F. E. Human fibronectin: molecular cloning evidence for two mRNA species differing by an internal segment coding for a structural domain. EMBO J. 1984 Jan;3(1):221–226. doi: 10.1002/j.1460-2075.1984.tb01787.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Matsuura H., Hakomori S. The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6: its presence in fibronectins from fetal and tumor tissues and its absence in those from normal adult tissues and plasma. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6517–6521. doi: 10.1073/pnas.82.19.6517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Owens R. J., Kornblihtt A. R., Baralle F. E. Fibronectin, the generation of multiple polypeptides from a single gene. Oxf Surv Eukaryot Genes. 1986;3:141–160. [PubMed] [Google Scholar]
  18. Paul J. I., Schwarzbauer J. E., Tamkun J. W., Hynes R. O. Cell-type-specific fibronectin subunits generated by alternative splicing. J Biol Chem. 1986 Sep 15;261(26):12258–12265. [PubMed] [Google Scholar]
  19. Petersen T. E., Thøgersen H. C., Skorstengaard K., Vibe-Pedersen K., Sahl P., Sottrup-Jensen L., Magnusson S. Partial primary structure of bovine plasma fibronectin: three types of internal homology. Proc Natl Acad Sci U S A. 1983 Jan;80(1):137–141. doi: 10.1073/pnas.80.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pierschbacher M. D., Hayman E. G., Ruoslahti E. Location of the cell-attachment site in fibronectin with monoclonal antibodies and proteolytic fragments of the molecule. Cell. 1981 Oct;26(2 Pt 2):259–267. doi: 10.1016/0092-8674(81)90308-1. [DOI] [PubMed] [Google Scholar]
  21. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  22. Schwarzbauer J. E., Paul J. I., Hynes R. O. On the origin of species of fibronectin. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1424–1428. doi: 10.1073/pnas.82.5.1424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sekiguchi K., Hakomori S. Domain structure of human plasma fibronectin. Differences and similarities between human and hamster fibronectins. J Biol Chem. 1983 Mar 25;258(6):3967–3973. [PubMed] [Google Scholar]
  24. Sekiguchi K., Siri A., Zardi L., Hakomori S. Differences in domain structure between human fibronectins isolated from plasma and from culture supernatants of normal and transformed fibroblasts. Studies with domain-specific antibodies. J Biol Chem. 1985 Apr 25;260(8):5105–5114. [PubMed] [Google Scholar]
  25. Skorstengaard K., Jensen M. S., Sahl P., Petersen T. E., Magnusson S. Complete primary structure of bovine plasma fibronectin. Eur J Biochem. 1986 Dec 1;161(2):441–453. doi: 10.1111/j.1432-1033.1986.tb10464.x. [DOI] [PubMed] [Google Scholar]
  26. Tamkun J. W., Hynes R. O. Plasma fibronectin is synthesized and secreted by hepatocytes. J Biol Chem. 1983 Apr 10;258(7):4641–4647. [PubMed] [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vibe-Pedersen K., Kornblihtt A. R., Baralle F. E. Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing. EMBO J. 1984 Nov;3(11):2511–2516. doi: 10.1002/j.1460-2075.1984.tb02165.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yamada K. M. Cell surface interactions with extracellular materials. Annu Rev Biochem. 1983;52:761–799. doi: 10.1146/annurev.bi.52.070183.003553. [DOI] [PubMed] [Google Scholar]
  30. Zardi L., Carnemolla B., Balza E., Borsi L., Castellani P., Rocco M., Siri A. Elution of fibronectin proteolytic fragments from a hydroxyapatite chromatography column. A simple procedure for the purification of fibronectin domains. Eur J Biochem. 1985 Feb 1;146(3):571–579. doi: 10.1111/j.1432-1033.1985.tb08690.x. [DOI] [PubMed] [Google Scholar]
  31. Zardi L., Carnemolla B., Siri A., Santi L., Accolla R. S. Somatic cell hybrids producing antibodies specific to human fibronectin. Int J Cancer. 1980 Mar 15;25(3):325–329. doi: 10.1002/ijc.2910250304. [DOI] [PubMed] [Google Scholar]
  32. Zardi L., Siri A., Carnemolla B., Cosulich E., Viale G., Santi L. A simplified procedure for the preparation of antibodies to serum fibronectin. J Immunol Methods. 1980;34(2):155–165. doi: 10.1016/0022-1759(80)90169-6. [DOI] [PubMed] [Google Scholar]