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. 1987 Mar 1;104(3):595–600. doi: 10.1083/jcb.104.3.595

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells

PMCID: PMC2114534  PMID: 3643927

Abstract

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.

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Selected References

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  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. 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]
  4. Chang S. E., Keen J., Lane E. B., Taylor-Papadimitriou J. Establishment and characterization of SV40-transformed human breast epithelial cell lines. Cancer Res. 1982 May;42(5):2040–2053. [PubMed] [Google Scholar]
  5. Hawke D. H., Harris D. C., Shively J. E. Microsequence analysis of peptides and proteins. V. Design and performance of a novel gas-liquid-solid phase instrument. Anal Biochem. 1985 Jun;147(2):315–330. doi: 10.1016/0003-2697(85)90278-7. [DOI] [PubMed] [Google Scholar]
  6. Hawke D., Yuan P. M., Shively J. E. Microsequence analysis of peptides and proteins. II. Separation of amino acid phenylthiohydantoin derivatives by high-performance liquid chromatography on octadecylsilane supports. Anal Biochem. 1982 Mar 1;120(2):302–311. doi: 10.1016/0003-2697(82)90351-7. [DOI] [PubMed] [Google Scholar]
  7. Hynes R. O., Yamada K. M. Fibronectins: multifunctional modular glycoproteins. J Cell Biol. 1982 Nov;95(2 Pt 1):369–377. doi: 10.1083/jcb.95.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Keen J., Chang S. E., Taylor-Papadimitriou J. Monoclonal antibodies that distinguish between human cellular and plasma fibronectin. Mol Biol Med. 1984 Feb;2(1):15–27. [PubMed] [Google Scholar]
  11. Koch G. A., Schoen R. C., Klebe R. J., Shows T. B. Assignment of a fibronection gene to human chromosome 2 using monoclonal antibodies. Exp Cell Res. 1982 Oct;141(2):293–302. doi: 10.1016/0014-4827(82)90217-8. [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. Kornblihtt A. R., Vibe-Pedersen K., Baralle F. E. Isolation and characterization of cDNA clones for human and bovine fibronectins. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3218–3222. doi: 10.1073/pnas.80.11.3218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Mosesson M. W., Umfleet R. A. The cold-insoluble globulin of human plasma. I. Purification, primary characterization, and relationship to fibrinogen and other cold-insoluble fraction components. J Biol Chem. 1970 Nov 10;245(21):5728–5736. [PubMed] [Google Scholar]
  18. Mosher D. F. Physiology of fibronectin. Annu Rev Med. 1984;35:561–575. doi: 10.1146/annurev.me.35.020184.003021. [DOI] [PubMed] [Google Scholar]
  19. Pande H., Calaycay J., Hawke D., Ben-Avram C. M., Shively J. E. Primary structure of a glycosylated DNA-binding domain in human plasma fibronectin. J Biol Chem. 1985 Feb 25;260(4):2301–2306. [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Ruoslahti E., Engvall E., Hayman E. G. Fibronectin: current concepts of its structure and functions. Coll Relat Res. 1981;1(1):95–128. doi: 10.1016/s0174-173x(80)80011-2. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Schwarzbauer J. E., Tamkun J. W., Lemischka I. R., Hynes R. O. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell. 1983 Dec;35(2 Pt 1):421–431. doi: 10.1016/0092-8674(83)90175-7. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Tamkun J. W., Schwarzbauer J. E., Hynes R. O. A single rat fibronectin gene generates three different mRNAs by alternative splicing of a complex exon. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5140–5144. doi: 10.1073/pnas.81.16.5140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  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., Cianfriglia M., Balza E., Carnemolla B., Siri A., Croce C. M. Species-specific monoclonal antibodies in the assignment of the gene for human fibronectin to chromosome 2. EMBO J. 1982;1(8):929–933. doi: 10.1002/j.1460-2075.1982.tb01273.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Zardi L., Siri A., Carnemolla B., Santi L., Gardner W. D., Hoch S. O. Fibronectin: a chromatin-associated protein? Cell. 1979 Nov;18(3):649–657. doi: 10.1016/0092-8674(79)90120-x. [DOI] [PubMed] [Google Scholar]

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