Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Aug 1;109(2):843–852. doi: 10.1083/jcb.109.2.843

Expression and analysis of COOH-terminal deletions of the human thrombospondin molecule

PMCID: PMC2115704  PMID: 2760115

Abstract

Thrombospondin (TSP) is a homotrimeric extracellular glycoprotein with a subunit molecular mass of 140 kD. The subunits have a modular or domain-like structure and are held together by interchain disulphide bonds. A number of domains have been identified including those for the binding of collagen, fibrinogen, and heparin. Due to the trimeric form of the TSP molecule, the various domains are trivalent in nature and this contributes to the ability of TSP to mediate cell-substrate interactions. Indeed, TSP has recently been shown not only to promote cell adhesion but also to be intimately involved in cell growth and migration. The adhesive function of TSP is attributable to the "solid- phase" or matrix-bound form of the molecule. There is some evidence that the heparin-binding domain mediates incorporation of soluble TSP into the insoluble matrix form. The heparin-binding domain of TSP is a compact globular amino-terminal moiety that contains two clusters of basic amino acids and a single intrachain disulphide bond. To delineate the role of the heparin-binding domain in matrix assembly and to define further the precise region of interchain disulphide bonding that results in trimer formation, we have expressed deleted forms of the cDNA encoding TSP in SV-40-transformed. African green monkey kidney cells. The proteins synthesized from the various deleted TSP cDNAs were examined for (a) secretion into the culture medium and incorporation into the extracellular matrix; (b) binding to heparin-Sepharose; (c) immunoprecipitability by a conformation-specific monoclonal antibody; and (d) ability to form trimers. This analysis allowed us to draw the following conclusions. (a) A 218 amino acid NH2-terminal protein that preserves the intrachain disulphide bridge of the heparin-binding domain is capable of binding to heparin-Sepharose and incorporating into the extracellular matrix. (b) A shorter 164 amino acid NH2- terminal peptide that does not contain the intrachain disulphide bridge of the heparin-binding domain is neither able to bind to heparin- Sepharose nor able to incorporate into the extracellular matrix. (c) The region of interchain disulphide bridging necessary for trimer assembly resides within a cluster of seven cysteine residues immediately adjacent to the heparin-binding domain.

Full Text

The Full Text of this article is available as a PDF (2.8 MB).

Selected References

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

  1. Baenziger N. L., Brodie G. N., Majerus P. W. Isolation and properties of a thrombin-sensitive protein of human platelets. J Biol Chem. 1972 May 10;247(9):2723–2731. [PubMed] [Google Scholar]
  2. Dixit V. M., Galvin N. J., O'Rourke K. M., Frazier W. A. Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes. J Biol Chem. 1986 Feb 5;261(4):1962–1968. [PubMed] [Google Scholar]
  3. Dixit V. M., Grant G. A., Santoro S. A., Frazier W. A. Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin. J Biol Chem. 1984 Aug 25;259(16):10100–10105. [PubMed] [Google Scholar]
  4. Dixit V. M., Haverstick D. M., O'Rourke K. M., Hennessy S. W., Grant G. A., Santoro S. A., Frazier W. A. A monoclonal antibody against human thrombospondin inhibits platelet aggregation. Proc Natl Acad Sci U S A. 1985 May;82(10):3472–3476. doi: 10.1073/pnas.82.10.3472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dixit V. M., Haverstick D. M., O'Rourke K. M., Hennessy S. W., Grant G. A., Santoro S. A., Frazier W. A. Effects of anti-thrombospondin monoclonal antibodies on the agglutination of erythrocytes and fixed, activated platelets by purified thrombospondin. Biochemistry. 1985 Jul 30;24(16):4270–4275. doi: 10.1021/bi00337a003. [DOI] [PubMed] [Google Scholar]
  6. Dixit V. M., Hennessy S. W., Grant G. A., Rotwein P., Frazier W. A. Characterization of a cDNA encoding the heparin and collagen binding domains of human thrombospondin. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5449–5453. doi: 10.1073/pnas.83.15.5449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Doyle C., Roth M. G., Sambrook J., Gething M. J. Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport. J Cell Biol. 1985 Mar;100(3):704–714. doi: 10.1083/jcb.100.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doyle C., Sambrook J., Gething M. J. Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin. J Cell Biol. 1986 Oct;103(4):1193–1204. doi: 10.1083/jcb.103.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frazier W. A. Thrombospondin: a modular adhesive glycoprotein of platelets and nucleated cells. J Cell Biol. 1987 Aug;105(2):625–632. doi: 10.1083/jcb.105.2.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Galvin N. J., Dixit V. M., O'Rourke K. M., Santoro S. A., Grant G. A., Frazier W. A. Mapping of epitopes for monoclonal antibodies against human platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing. J Cell Biol. 1985 Oct;101(4):1434–1441. doi: 10.1083/jcb.101.4.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Galvin N. J., Vance P. M., Dixit V. M., Fink B., Frazier W. A. Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy. J Cell Biol. 1987 May;104(5):1413–1422. doi: 10.1083/jcb.104.5.1413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  13. Gospodarowicz D., Ill C. R. Do plasma and serum have different abilities to promote cell growth? Proc Natl Acad Sci U S A. 1980 May;77(5):2726–2730. doi: 10.1073/pnas.77.5.2726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haverstick D. M., Dixit V. M., Grant G. A., Frazier W. A., Santoro S. A. Characterization of the platelet agglutinating activity of thrombospondin. Biochemistry. 1985 Jun 18;24(13):3128–3134. doi: 10.1021/bi00334a009. [DOI] [PubMed] [Google Scholar]
  15. Haverstick D. M., Dixit V. M., Grant G. A., Frazier W. A., Santoro S. A. Localization of the hemagglutinating activity of platelet thrombospondin to a 140 000-dalton thermolytic fragment. Biochemistry. 1984 Nov 6;23(23):5597–5603. doi: 10.1021/bi00318a033. [DOI] [PubMed] [Google Scholar]
  16. Hunt L. T., Barker W. C. von Willebrand factor shares a distinctive cysteine-rich domain with thrombospondin and procollagen. Biochem Biophys Res Commun. 1987 Apr 29;144(2):876–882. doi: 10.1016/s0006-291x(87)80046-3. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kobayashi S., Eden-McCutchan F., Framson P., Bornstein P. Partial amino acid sequence of human thrombospondin as determined by analysis of cDNA clones: homology to malarial circumsporozoite proteins. Biochemistry. 1986 Dec 30;25(26):8418–8425. doi: 10.1021/bi00374a014. [DOI] [PubMed] [Google Scholar]
  19. Lahav J., Lawler J., Gimbrone M. A. Thrombospondin interactions with fibronectin and fibrinogen. Mutual inhibition in binding. Eur J Biochem. 1984 Nov 15;145(1):151–156. doi: 10.1111/j.1432-1033.1984.tb08534.x. [DOI] [PubMed] [Google Scholar]
  20. Lahav J., Schwartz M. A., Hynes R. O. Analysis of platelet adhesion with a radioactive chemical crosslinking reagent: interaction of thrombospondin with fibronectin and collagen. Cell. 1982 Nov;31(1):253–262. doi: 10.1016/0092-8674(82)90425-1. [DOI] [PubMed] [Google Scholar]
  21. Lawler J., Hynes R. O. The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins. J Cell Biol. 1986 Nov;103(5):1635–1648. doi: 10.1083/jcb.103.5.1635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lawler J. The structural and functional properties of thrombospondin. Blood. 1986 May;67(5):1197–1209. [PubMed] [Google Scholar]
  23. Leung L. L. Role of thrombospondin in platelet aggregation. J Clin Invest. 1984 Nov;74(5):1764–1772. doi: 10.1172/JCI111595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Majack R. A., Cook S. C., Bornstein P. Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9050–9054. doi: 10.1073/pnas.83.23.9050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Majack R. A., Cook S. C., Bornstein P. Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells. J Cell Biol. 1985 Sep;101(3):1059–1070. doi: 10.1083/jcb.101.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Majack R. A., Goodman L. V., Dixit V. M. Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation. J Cell Biol. 1988 Feb;106(2):415–422. doi: 10.1083/jcb.106.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Majack R. A., Mildbrandt J., Dixit V. M. Induction of thrombospondin messenger RNA levels occurs as an immediate primary response to platelet-derived growth factor. J Biol Chem. 1987 Jun 25;262(18):8821–8825. [PubMed] [Google Scholar]
  28. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McKeown-Longo P. J., Hanning R., Mosher D. F. Binding and degradation of platelet thrombospondin by cultured fibroblasts. J Cell Biol. 1984 Jan;98(1):22–28. doi: 10.1083/jcb.98.1.22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mumby S. M., Raugi G. J., Bornstein P. Interactions of thrombospondin with extracellular matrix proteins: selective binding to type V collagen. J Cell Biol. 1984 Feb;98(2):646–652. doi: 10.1083/jcb.98.2.646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Murphy-Ullrich J. E., Mosher D. F. Interactions of thrombospondin with endothelial cells: receptor-mediated binding and degradation. J Cell Biol. 1987 Oct;105(4):1603–1611. doi: 10.1083/jcb.105.4.1603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Murphy-Ullrich J. E., Westrick L. G., Esko J. D., Mosher D. F. Altered metabolism of thrombospondin by Chinese hamster ovary cells defective in glycosaminoglycan synthesis. J Biol Chem. 1988 May 5;263(13):6400–6406. [PubMed] [Google Scholar]
  33. Prochownik E. V., Kukowska J. Deregulated expression of c-myc by murine erythroleukaemia cells prevents differentiation. 1986 Aug 28-Sep 3Nature. 322(6082):848–850. doi: 10.1038/322848a0. [DOI] [PubMed] [Google Scholar]
  34. Roberts D. D., Sherwood J. A., Ginsburg V. Platelet thrombospondin mediates attachment and spreading of human melanoma cells. J Cell Biol. 1987 Jan;104(1):131–139. doi: 10.1083/jcb.104.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tuszynski G. P., Rothman V., Murphy A., Siegler K., Smith L., Smith S., Karczewski J., Knudsen K. A. Thrombospondin promotes cell-substratum adhesion. Science. 1987 Jun 19;236(4808):1570–1573. doi: 10.1126/science.2438772. [DOI] [PubMed] [Google Scholar]
  36. Varani J., Dixit V. M., Fligiel S. E., McKeever P. E., Carey T. E. Thrombospondin-induced attachment and spreading of human squamous carcinoma cells. Exp Cell Res. 1986 Dec;167(2):376–390. doi: 10.1016/0014-4827(86)90178-3. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES