Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Oct 1;375(Pt 1):131–139. doi: 10.1042/BJ20030730

Binding of endostatin to endothelial heparan sulphate shows a differential requirement for specific sulphates.

Fiona H Blackhall 1, Catherine L R Merry 1, Malcolm Lyon 1, Gordon C Jayson 1, Judah Folkman 1, Kashi Javaherian 1, John T Gallagher 1
PMCID: PMC1223655  PMID: 12812520

Abstract

Endostatin is a naturally occurring proteolytic fragment of the C-terminal domain of collagen XVIII. It inhibits angiogenesis by a mechanism that appears to involve binding to HS (heparan sulphate). We have examined the molecular interaction between endostatin and HS from micro- and macrovessel endothelial cells. Two discrete panels of oligosaccharides were prepared from metabolically radiolabelled HS, using digestion with either heparinase I or III, and then examined for their endostatin affinity using a sensitive filter-binding assay. Two types of endostatin-binding regions were identified: one comprising sulphated domains of five or more disaccharides in length, enriched in 6-O-sulphate groups, and the other contained long heparinase I-resistant fragments. In the latter case, evidence from the present study suggests that the binding region encompasses a sulphated domain fragment and a transition zone of intermediate sulphation. The contribution to binding of specific O-sulphate groups was determined using selectively desulphated HS species, namely HS from Hs2st-/- mutant cells, and by comparing the compositions of endostatin-binding and non-binding oligosaccharides. The results indicate that 6-O-sulphates play a dominant role in site selectivity and 2-O-sulphates are not strictly essential.

Full Text

The Full Text of this article is available as a PDF (180.1 KB).

Selected References

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

  1. Bernfield M., Götte M., Park P. W., Reizes O., Fitzgerald M. L., Lincecum J., Zako M. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem. 1999;68:729–777. doi: 10.1146/annurev.biochem.68.1.729. [DOI] [PubMed] [Google Scholar]
  2. Dhanabal M., Ramchandran R., Waterman M. J., Lu H., Knebelmann B., Segal M., Sukhatme V. P. Endostatin induces endothelial cell apoptosis. J Biol Chem. 1999 Apr 23;274(17):11721–11726. doi: 10.1074/jbc.274.17.11721. [DOI] [PubMed] [Google Scholar]
  3. Ding Y. H., Javaherian K., Lo K. M., Chopra R., Boehm T., Lanciotti J., Harris B. A., Li Y., Shapiro R., Hohenester E. Zinc-dependent dimers observed in crystals of human endostatin. Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10443–10448. doi: 10.1073/pnas.95.18.10443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dixelius J., Larsson H., Sasaki T., Holmqvist K., Lu L., Engström A., Timpl R., Welsh M., Claesson-Welsh L. Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis. Blood. 2000 Jun 1;95(11):3403–3411. [PubMed] [Google Scholar]
  5. Halfter W., Dong S., Schurer B., Cole G. J. Collagen XVIII is a basement membrane heparan sulfate proteoglycan. J Biol Chem. 1998 Sep 25;273(39):25404–25412. doi: 10.1074/jbc.273.39.25404. [DOI] [PubMed] [Google Scholar]
  6. Hohenester E., Sasaki T., Olsen B. R., Timpl R. Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution. EMBO J. 1998 Mar 16;17(6):1656–1664. doi: 10.1093/emboj/17.6.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Karihaloo A., Karumanchi S. A., Barasch J., Jha V., Nickel C. H., Yang J., Grisaru S., Bush K. T., Nigam S., Rosenblum N. D. Endostatin regulates branching morphogenesis of renal epithelial cells and ureteric bud. Proc Natl Acad Sci U S A. 2001 Oct 16;98(22):12509–12514. doi: 10.1073/pnas.221205198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Karumanchi S. A., Jha V., Ramchandran R., Karihaloo A., Tsiokas L., Chan B., Dhanabal M., Hanai J. I., Venkataraman G., Shriver Z. Cell surface glypicans are low-affinity endostatin receptors. Mol Cell. 2001 Apr;7(4):811–822. doi: 10.1016/s1097-2765(01)00225-8. [DOI] [PubMed] [Google Scholar]
  9. Kreuger Johan, Matsumoto Taro, Vanwildemeersch Maarten, Sasaki Takako, Timpl Rupert, Claesson-Welsh Lena, Spillmann Dorothe, Lindahl Ulf. Role of heparan sulfate domain organization in endostatin inhibition of endothelial cell function. EMBO J. 2002 Dec 2;21(23):6303–6311. doi: 10.1093/emboj/cdf638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lindahl U., Kusche-Gullberg M., Kjellén L. Regulated diversity of heparan sulfate. J Biol Chem. 1998 Sep 25;273(39):24979–24982. doi: 10.1074/jbc.273.39.24979. [DOI] [PubMed] [Google Scholar]
  11. Lo K. M., Sudo Y., Chen J., Li Y., Lan Y., Kong S. M., Chen L., An Q., Gillies S. D. High level expression and secretion of Fc-X fusion proteins in mammalian cells. Protein Eng. 1998 Jun;11(6):495–500. doi: 10.1093/protein/11.6.495. [DOI] [PubMed] [Google Scholar]
  12. Lyon M., Deakin J. A., Mizuno K., Nakamura T., Gallagher J. T. Interaction of hepatocyte growth factor with heparan sulfate. Elucidation of the major heparan sulfate structural determinants. J Biol Chem. 1994 Apr 15;269(15):11216–11223. [PubMed] [Google Scholar]
  13. Lyon M., Rushton G., Askari J. A., Humphries M. J., Gallagher J. T. Elucidation of the structural features of heparan sulfate important for interaction with the Hep-2 domain of fibronectin. J Biol Chem. 2000 Feb 18;275(7):4599–4606. doi: 10.1074/jbc.275.7.4599. [DOI] [PubMed] [Google Scholar]
  14. Maccarana M., Lindahl U. Mode of interaction between platelet factor 4 and heparin. Glycobiology. 1993 Jun;3(3):271–277. doi: 10.1093/glycob/3.3.271. [DOI] [PubMed] [Google Scholar]
  15. Merry C. L., Bullock S. L., Swan D. C., Backen A. C., Lyon M., Beddington R. S., Wilson V. A., Gallagher J. T. The molecular phenotype of heparan sulfate in the Hs2st-/- mutant mouse. J Biol Chem. 2001 Jul 16;276(38):35429–35434. doi: 10.1074/jbc.M100379200. [DOI] [PubMed] [Google Scholar]
  16. Merry C. L., Lyon M., Deakin J. A., Hopwood J. J., Gallagher J. T. Highly sensitive sequencing of the sulfated domains of heparan sulfate. J Biol Chem. 1999 Jun 25;274(26):18455–18462. doi: 10.1074/jbc.274.26.18455. [DOI] [PubMed] [Google Scholar]
  17. O'Reilly M. S., Boehm T., Shing Y., Fukai N., Vasios G., Lane W. S., Flynn E., Birkhead J. R., Olsen B. R., Folkman J. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997 Jan 24;88(2):277–285. doi: 10.1016/s0092-8674(00)81848-6. [DOI] [PubMed] [Google Scholar]
  18. Pye D. A., Vives R. R., Turnbull J. E., Hyde P., Gallagher J. T. Heparan sulfate oligosaccharides require 6-O-sulfation for promotion of basic fibroblast growth factor mitogenic activity. J Biol Chem. 1998 Sep 4;273(36):22936–22942. doi: 10.1074/jbc.273.36.22936. [DOI] [PubMed] [Google Scholar]
  19. Rice K. G., Linhardt R. J. Study of structurally defined oligosaccharide substrates of heparin and heparan monosulfate lyases. Carbohydr Res. 1989 Jul 15;190(2):219–233. doi: 10.1016/0008-6215(89)84127-8. [DOI] [PubMed] [Google Scholar]
  20. Salmivirta M., Lidholt K., Lindahl U. Heparan sulfate: a piece of information. FASEB J. 1996 Sep;10(11):1270–1279. doi: 10.1096/fasebj.10.11.8836040. [DOI] [PubMed] [Google Scholar]
  21. Sasaki T., Fukai N., Mann K., Göhring W., Olsen B. R., Timpl R. Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO J. 1998 Aug 3;17(15):4249–4256. doi: 10.1093/emboj/17.15.4249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sasaki T., Larsson H., Kreuger J., Salmivirta M., Claesson-Welsh L., Lindahl U., Hohenester E., Timpl R. Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin. EMBO J. 1999 Nov 15;18(22):6240–6248. doi: 10.1093/emboj/18.22.6240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Taddei L., Chiarugi P., Brogelli L., Cirri P., Magnelli L., Raugei G., Ziche M., Granger H. J., Chiarugi V., Ramponi G. Inhibitory effect of full-length human endostatin on in vitro angiogenesis. Biochem Biophys Res Commun. 1999 Sep 24;263(2):340–345. doi: 10.1006/bbrc.1999.1342. [DOI] [PubMed] [Google Scholar]
  24. Turnbull J. E., Fernig D. G., Ke Y., Wilkinson M. C., Gallagher J. T. Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate. J Biol Chem. 1992 May 25;267(15):10337–10341. [PubMed] [Google Scholar]
  25. Turnbull J. E., Gallagher J. T. Sequence analysis of heparan sulphate indicates defined location of N-sulphated glucosamine and iduronate 2-sulphate residues proximal to the protein-linkage region. Biochem J. 1991 Jul 15;277(Pt 2):297–303. doi: 10.1042/bj2770297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wasteson A. A method for the determination of the molecular weight and molecular-weight distribution of chondroitin sulphate. J Chromatogr. 1971 Jul 8;59(1):87–97. doi: 10.1016/s0021-9673(01)80009-1. [DOI] [PubMed] [Google Scholar]
  27. Yamada S., Yoshida K., Sugiura M., Sugahara K., Khoo K. H., Morris H. R., Dell A. Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin. J Biol Chem. 1993 Mar 5;268(7):4780–4787. [PubMed] [Google Scholar]
  28. Yamaguchi N., Anand-Apte B., Lee M., Sasaki T., Fukai N., Shapiro R., Que I., Lowik C., Timpl R., Olsen B. R. Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding. EMBO J. 1999 Aug 16;18(16):4414–4423. doi: 10.1093/emboj/18.16.4414. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES