Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Sep 1;342(Pt 2):361–368.

Evidence that platelet and tumour heparanases are similar enzymes.

C Freeman 1, A M Browne 1, C R Parish 1
PMCID: PMC1220473  PMID: 10455023

Abstract

In order to enter tissues, blood-borne metastatic tumour cells and leucocytes need to extravasate through the vascular basal lamina (BL), a process which involves a battery of degradative enzymes. A key degradative enzyme is the endoglycosidase heparanase, which cleaves heparan sulphate (HS), an important structural component of the vascular BL. Previously, tumour-derived heparanase activity (which has been shown to be related to the metastatic potential of murine and human melanoma cell lines) was reported to cleave HS and be inhibited by heparin, as distinct from human platelet heparanase, which cleaved both substrates [Nakajima, Irimura and Nicolson (1988) J. Cell Biochem. 36, 157-167]. We recently reported the purification of human platelet heparanase and showed that the enzyme is a 50-kDa endoglucuronidase [Freeman and Parish (1998) Biochem. J. 330, 1341-1350]. We now report the purification and characterization of heparanase activity from highly metastatic rat 13762 MAT mammary adenocarcinoma and human HCT 116 colonic carcinoma cells and from rat liver using essentially the same procedure that was reported for purification of the human platelet enzyme. The rat 13762 MAT tumour enzyme, which has a native M(r) of 45 kDa when analysed by gel-filtration chromatography and by SDS/PAGE, was observed to be an endoglucuronidase that degraded heparin and HS to fragments of the same sizes as the human platelet enzyme does. N-deglycosylation of both the human platelet and rat 13762 MAT tumour enzymes gave, in each case, a 41-kDa band by SDS/PAGE analysis, demonstrating that the observed difference in M(r) between the platelet and tumour enzymes may have been due largely to differences in the relative amounts of N-glycosylation. Two peptides were isolated following Endoproteinase Lys-C digestion of both the human platelet and rat 13762 MAT tumour heparanases and were shown to be highly similar. Both the rat liver and human colonic carcinoma heparanases also degraded both heparin and HS to fragments of the same sizes as the human platelet enzyme does. Western-blot analysis of an SDS/PAGE gel using antibodies raised against human platelet heparanase demonstrated that human platelet, human tumour and rat tumour heparanases were immunochemically cross-reactive. In conclusion, because of the similarities in their sizes, substrate specificities, peptide sequences and immunoreactivities, we propose that heparanase activities present in human platelets, rat liver and in rat and human tumour cells are, in fact, mediated by a similar enzyme.

Full Text

The Full Text of this article is available as a PDF (155.9 KB).

Selected References

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

  1. Arbogast B., Hopwood J. J., Dorfman A. Heparinase activity in rat liver. Biochem Biophys Res Commun. 1977 Apr 11;75(3):610–617. doi: 10.1016/0006-291x(77)91516-9. [DOI] [PubMed] [Google Scholar]
  2. Bame K. J., Hassall A., Sanderson C., Venkatesan I., Sun C. Partial purification of heparanase activities in Chinese hamster ovary cells: evidence for multiple intracellular heparanases. Biochem J. 1998 Nov 15;336(Pt 1):191–200. doi: 10.1042/bj3360191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bame K. J., Robson K. Heparanases produce distinct populations of heparan sulfate glycosaminoglycans in Chinese hamster ovary cells. J Biol Chem. 1997 Jan 24;272(4):2245–2251. [PubMed] [Google Scholar]
  4. Bartlett M. R., Underwood P. A., Parish C. R. Comparative analysis of the ability of leucocytes, endothelial cells and platelets to degrade the subendothelial basement membrane: evidence for cytokine dependence and detection of a novel sulfatase. Immunol Cell Biol. 1995 Apr;73(2):113–124. doi: 10.1038/icb.1995.19. [DOI] [PubMed] [Google Scholar]
  5. Campbell J. H., Rennick R. E., Kalevitch S. G., Campbell G. R. Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype. Exp Cell Res. 1992 May;200(1):156–167. doi: 10.1016/s0014-4827(05)80084-9. [DOI] [PubMed] [Google Scholar]
  6. Clements P. R., Brooks D. A., McCourt P. A., Hopwood J. J. Immunopurification and characterization of human alpha-L-iduronidase with the use of monoclonal antibodies. Biochem J. 1989 Apr 1;259(1):199–208. doi: 10.1042/bj2590199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Vouge M. W., Yamazaki A., Bennett S. A., Chen J. H., Shwed P. S., Couture C., Birnboim H. C. Immunoselection of GRP94/endoplasmin from a KNRK cell-specific lambda gt11 library using antibodies directed against a putative heparanase amino-terminal peptide. Int J Cancer. 1994 Jan 15;56(2):286–294. doi: 10.1002/ijc.2910560224. [DOI] [PubMed] [Google Scholar]
  8. Freeman C., Hopwood J. Lysosomal degradation of heparin and heparan sulphate. Adv Exp Med Biol. 1992;313:121–134. doi: 10.1007/978-1-4899-2444-5_13. [DOI] [PubMed] [Google Scholar]
  9. Freeman C., Parish C. R. A rapid quantitative assay for the detection of mammalian heparanase activity. Biochem J. 1997 Jul 1;325(Pt 1):229–237. doi: 10.1042/bj3250229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Freeman C., Parish C. R. Human platelet heparanase: purification, characterization and catalytic activity. Biochem J. 1998 Mar 15;330(Pt 3):1341–1350. doi: 10.1042/bj3301341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gilat D., Hershkoviz R., Goldkorn I., Cahalon L., Korner G., Vlodavsky I., Lider O. Molecular behavior adapts to context: heparanase functions as an extracellular matrix-degrading enzyme or as a T cell adhesion molecule, depending on the local pH. J Exp Med. 1995 May 1;181(5):1929–1934. doi: 10.1084/jem.181.5.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Godder K., Vlodavsky I., Eldor A., Weksler B. B., Haimovitz-Freidman A., Fuks Z. Heparanase activity in cultured endothelial cells. J Cell Physiol. 1991 Aug;148(2):274–280. doi: 10.1002/jcp.1041480213. [DOI] [PubMed] [Google Scholar]
  13. Gonzalez-Stawinski G. V., Parker W., Holzknecht Z. E., Huber N. S., Platt J. L. Partial sequence of human platelet heparitinase and evidence of its ability to polymerize. Biochim Biophys Acta. 1999 Jan 11;1429(2):431–438. doi: 10.1016/s0167-4838(98)00254-4. [DOI] [PubMed] [Google Scholar]
  14. Goshen R., Hochberg A. A., Korner G., Levy E., Ishai-Michaeli R., Elkin M., de Groot N., Vlodavsky I. Purification and characterization of placental heparanase and its expression by cultured cytotrophoblasts. Mol Hum Reprod. 1996 Sep;2(9):679–684. doi: 10.1093/molehr/2.9.679. [DOI] [PubMed] [Google Scholar]
  15. Graham L. D. Tumour rejection antigens of the hsp90 family (gp96) closely resemble tumour-associated heparanase enzymes. Biochem J. 1994 Aug 1;301(Pt 3):917–918. doi: 10.1042/bj3010917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Graham L. D., Underwood P. A. Comparison of the heparanase enzymes from mouse melanoma cells, mouse macrophages, and human platelets. Biochem Mol Biol Int. 1996 Jun;39(3):563–571. doi: 10.1080/15216549600201621. [DOI] [PubMed] [Google Scholar]
  17. Hellman U., Wernstedt C., Góez J., Heldin C. H. Improvement of an "In-Gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal Biochem. 1995 Jan 1;224(1):451–455. doi: 10.1006/abio.1995.1070. [DOI] [PubMed] [Google Scholar]
  18. Hoogewerf A. J., Leone J. W., Reardon I. M., Howe W. J., Asa D., Heinrikson R. L., Ledbetter S. R. CXC chemokines connective tissue activating peptide-III and neutrophil activating peptide-2 are heparin/heparan sulfate-degrading enzymes. J Biol Chem. 1995 Feb 17;270(7):3268–3277. doi: 10.1074/jbc.270.7.3268. [DOI] [PubMed] [Google Scholar]
  19. Hulett M. D., Freeman C., Hamdorf B. J., Baker R. T., Harris M. J., Parish C. R. Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis. Nat Med. 1999 Jul;5(7):803–809. doi: 10.1038/10525. [DOI] [PubMed] [Google Scholar]
  20. Ihrcke N. S., Parker W., Reissner K. J., Platt J. L. Regulation of platelet heparanase during inflammation: role of pH and proteinases. J Cell Physiol. 1998 Jun;175(3):255–267. doi: 10.1002/(SICI)1097-4652(199806)175:3<255::AID-JCP3>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
  21. Irimura T., Nakajima M., Nicolson G. L. Chemically modified heparins as inhibitors of heparan sulfate specific endo-beta-glucuronidase (heparanase) of metastatic melanoma cells. Biochemistry. 1986 Sep 9;25(18):5322–5328. doi: 10.1021/bi00366a050. [DOI] [PubMed] [Google Scholar]
  22. Jin L., Nakajima M., Nicolson G. L. Immunochemical localization of heparanase in mouse and human melanomas. Int J Cancer. 1990 Jun 15;45(6):1088–1095. doi: 10.1002/ijc.2910450618. [DOI] [PubMed] [Google Scholar]
  23. Kjellén L., Pertoft H., Oldberg A., Hök M. Oligosaccharides generated by an endoglucuronidase are intermediates in the intracellular degradation of heparan sulfate proteoglycans. J Biol Chem. 1985 Jul 15;260(14):8416–8422. [PubMed] [Google Scholar]
  24. Klein U., Kresse H., von Figura K. Evidence for degradation of heparan sulfate by endoglycosidases: glucosamine and hexuronic acid are reducing terminals of intracellular heparan sulfate from human skin fibroblasts. Biochem Biophys Res Commun. 1976 Mar 8;69(1):158–166. doi: 10.1016/s0006-291x(76)80286-0. [DOI] [PubMed] [Google Scholar]
  25. Klein U., Von Figura K. Partial purification and characterization of heparan sulfate specific endoglucuronidase. Biochem Biophys Res Commun. 1976 Dec 6;73(3):569–576. doi: 10.1016/0006-291x(76)90848-2. [DOI] [PubMed] [Google Scholar]
  26. Klein U., von Figura K. Substrate specificity of a heparan sulfate-degrading endoglucuronidase from human placenta. Hoppe Seylers Z Physiol Chem. 1979 Oct;360(10):1465–1471. doi: 10.1515/bchm2.1979.360.2.1465. [DOI] [PubMed] [Google Scholar]
  27. Kosir M. A., Quinn C. C., Zukowski K. L., Grignon D. J., Ledbetter S. Human prostate carcinoma cells produce extracellular heparanase. J Surg Res. 1997 Jan;67(1):98–105. doi: 10.1006/jsre.1996.4976. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Lapierre F., Holme K., Lam L., Tressler R. J., Storm N., Wee J., Stack R. J., Castellot J., Tyrrell D. J. Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase-inhibitory, angiostatic, anti-tumor and anti-metastatic properties. Glycobiology. 1996 Apr;6(3):355–366. doi: 10.1093/glycob/6.3.355. [DOI] [PubMed] [Google Scholar]
  30. Larsen A. K., Lund D. P., Langer R., Folkman J. Oral heparin results in the appearance of heparin fragments in the plasma of rats. Proc Natl Acad Sci U S A. 1986 May;83(9):2964–2968. doi: 10.1073/pnas.83.9.2964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Laskov R., Michaeli R. I., Sharir H., Yefenof E., Vlodavsky I. Production of heparanase by normal and neoplastic murine B-lymphocytes. Int J Cancer. 1991 Jan 2;47(1):92–98. doi: 10.1002/ijc.2910470117. [DOI] [PubMed] [Google Scholar]
  32. Matzner Y., Bar-Ner M., Yahalom J., Ishai-Michaeli R., Fuks Z., Vlodavsky I. Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes. J Clin Invest. 1985 Oct;76(4):1306–1313. doi: 10.1172/JCI112104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nakajima M., Irimura T., Di Ferrante D., Di Ferrante N., Nicolson G. L. Heparan sulfate degradation: relation to tumor invasive and metastatic properties of mouse B16 melanoma sublines. Science. 1983 May 6;220(4597):611–613. doi: 10.1126/science.6220468. [DOI] [PubMed] [Google Scholar]
  34. Nakajima M., Irimura T., Di Ferrante N., Nicolson G. L. Metastatic melanoma cell heparanase. Characterization of heparan sulfate degradation fragments produced by B16 melanoma endoglucuronidase. J Biol Chem. 1984 Feb 25;259(4):2283–2290. [PubMed] [Google Scholar]
  35. Nakajima M., Irimura T., Nicolson G. L. A solid-phase substrate of heparanase: its application to assay of human melanoma for heparan sulfate degradative activity. Anal Biochem. 1986 Aug 15;157(1):162–171. doi: 10.1016/0003-2697(86)90209-5. [DOI] [PubMed] [Google Scholar]
  36. Nakajima M., Irimura T., Nicolson G. L. Heparanases and tumor metastasis. J Cell Biochem. 1988 Feb;36(2):157–167. doi: 10.1002/jcb.240360207. [DOI] [PubMed] [Google Scholar]
  37. Nakajima M., Irimura T., Nicolson G. L. Tumor metastasis-associated heparanase (heparan sulfate endoglycosidase) activity in human melanoma cells. Cancer Lett. 1986 Jun;31(3):277–283. doi: 10.1016/0304-3835(86)90148-5. [DOI] [PubMed] [Google Scholar]
  38. Naparstek Y., Cohen I. R., Fuks Z., Vlodavsky I. Activated T lymphocytes produce a matrix-degrading heparan sulphate endoglycosidase. Nature. 1984 Jul 19;310(5974):241–244. doi: 10.1038/310241a0. [DOI] [PubMed] [Google Scholar]
  39. Oldberg A., Heldin C. H., Wasteson A., Busch C., Hök M. Characterization of a platelet endoglycosidase degrading heparin-like polysaccharides. Biochemistry. 1980 Dec 9;19(25):5755–5762. doi: 10.1021/bi00566a014. [DOI] [PubMed] [Google Scholar]
  40. Oosta G. M., Favreau L. V., Beeler D. L., Rosenberg R. D. Purification and properties of human platelet heparitinase. J Biol Chem. 1982 Oct 10;257(19):11249–11255. [PubMed] [Google Scholar]
  41. Parish C. R., Coombe D. R., Jakobsen K. B., Bennett F. A., Underwood P. A. Evidence that sulphated polysaccharides inhibit tumour metastasis by blocking tumour-cell-derived heparanases. Int J Cancer. 1987 Oct 15;40(4):511–518. doi: 10.1002/ijc.2910400414. [DOI] [PubMed] [Google Scholar]
  42. Parish C. R., Jakobsen K. B., Coombe D. R. A basement-membrane permeability assay which correlates with the metastatic potential of tumour cells. Int J Cancer. 1992 Sep 30;52(3):378–383. doi: 10.1002/ijc.2910520309. [DOI] [PubMed] [Google Scholar]
  43. Pikas D. S., Li J. P., Vlodavsky I., Lindahl U. Substrate specificity of heparanases from human hepatoma and platelets. J Biol Chem. 1998 Jul 24;273(30):18770–18777. doi: 10.1074/jbc.273.30.18770. [DOI] [PubMed] [Google Scholar]
  44. Ricoveri W., Cappelletti R. Heparan sulfate endoglycosidase and metastatic potential in murine fibrosarcoma and melanoma. Cancer Res. 1986 Aug;46(8):3855–3861. [PubMed] [Google Scholar]
  45. Schlechte W., Murano G., Boyd D. Examination of the role of the urokinase receptor in human colon cancer mediated laminin degradation. Cancer Res. 1989 Nov 1;49(21):6064–6069. [PubMed] [Google Scholar]
  46. Sewell R. F., Brenchley P. E., Mallick N. P. Human mononuclear cells contain an endoglycosidase specific for heparan sulphate glycosaminoglycan demonstrable with the use of a specific solid-phase metabolically radiolabelled substrate. Biochem J. 1989 Dec 15;264(3):777–783. doi: 10.1042/bj2640777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Srivastava P. K. Endo-beta-D-glucuronidase (heparanase) activity of heat-shock protein/tumour rejection antigen gp96. Biochem J. 1994 Aug 1;301(Pt 3):919–919. doi: 10.1042/bj3010919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Thunberg L., Bäckström G., Wasteson A., Robinson H. C., Ogren S., Lindahl U. Enzymatic depolymerization of heparin-related polysaccharides. Substrate specificities of mouse mastocytoma and human platelet endo-beta-D-glucuronidases. J Biol Chem. 1982 Sep 10;257(17):10278–10282. [PubMed] [Google Scholar]
  49. Turnbull J. E., Gallagher J. T. Distribution of iduronate 2-sulphate residues in heparan sulphate. Evidence for an ordered polymeric structure. Biochem J. 1991 Feb 1;273(Pt 3):553–559. doi: 10.1042/bj2730553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Turnbull J. E., Gallagher J. T. Molecular organization of heparan sulphate from human skin fibroblasts. Biochem J. 1990 Feb 1;265(3):715–724. doi: 10.1042/bj2650715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Vlodavsky I., Eldor A., Haimovitz-Friedman A., Matzner Y., Ishai-Michaeli R., Lider O., Naparstek Y., Cohen I. R., Fuks Z. Expression of heparanase by platelets and circulating cells of the immune system: possible involvement in diapedesis and extravasation. Invasion Metastasis. 1992;12(2):112–127. [PubMed] [Google Scholar]
  52. Vlodavsky I., Friedmann Y., Elkin M., Aingorn H., Atzmon R., Ishai-Michaeli R., Bitan M., Pappo O., Peretz T., Michal I. Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis. Nat Med. 1999 Jul;5(7):793–802. doi: 10.1038/10518. [DOI] [PubMed] [Google Scholar]
  53. Vlodavsky I., Mohsen M., Lider O., Svahn C. M., Ekre H. P., Vigoda M., Ishai-Michaeli R., Peretz T. Inhibition of tumor metastasis by heparanase inhibiting species of heparin. Invasion Metastasis. 1994;14(1-6):290–302. [PubMed] [Google Scholar]
  54. 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]
  55. Yanagishita M., Hascall V. C. Metabolism of proteoglycans in rat ovarian granulosa cell culture. Multiple intracellular degradative pathways and the effect of chloroquine. J Biol Chem. 1984 Aug 25;259(16):10270–10283. [PubMed] [Google Scholar]

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

RESOURCES