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. 2003 Jul 15;373(Pt 2):423–435. doi: 10.1042/BJ20030318

Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells.

Edward McKenzie 1, Kathryn Young 1, Margaret Hircock 1, James Bennett 1, Maina Bhaman 1, Robert Felix 1, Paul Turner 1, Alasdair Stamps 1, David McMillan 1, Giles Saville 1, Stanley Ng 1, Sean Mason 1, Daniel Snell 1, Darren Schofield 1, Haiping Gong 1, Reid Townsend 1, John Gallagher 1, Martin Page 1, Raj Parekh 1, Colin Stubberfield 1
PMCID: PMC1223510  PMID: 12713442

Abstract

The mammalian endoglycosidase heparanase (Hpa1) is primarily responsible for cleaving heparan sulphate proteoglycans (HSPGs) present on the basement membrane of cells and its potential for remodelling the extracellular matrix (ECM) could be important in embryonic development and tumour metastasis. Elevated expression of this enzyme has been implicated in various pathological processes including tumour cell proliferation, metastasis, inflammation and angiogenesis. The enzyme therefore represents a potential therapeutic target. Hpa1 protein is initially synthesized as an inactive 65 kDa proenzyme that is then believed to be subsequently activated by proteolytic cleavage to generate an active heterodimer of 8 and 50 kDa polypeptides. By analysis of a series of Hpa1 deletion proteins we confirm that the 8 kDa subunit is essential for enzyme activity. We present here for the first time an insect cell expression system used for the generation of large amounts of recombinant protein of high specific activity. Individual subunits were cloned into baculoviral secretory vectors and co-expressed in insect cells. Active secreted heterodimer protein was recovered from the medium and isolated by a one-step heparin-Sepharose chromatography procedure to give protein of >90% purity. The recombinant enzyme behaved similarly to the native protein with respect to the size of HS fragments liberated on digestion, substrate cleavage specificity and its preference for acidic pH. A significant amount of activity, however, was also detectable at physiological pH values, as measured both by an in vitro assay and by in vivo degradation of cell-bound heparan sulphate.

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Selected References

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  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. Cheng Fang, Mani Katrin, van den Born Jacob, Ding Kan, Belting Mattias, Fransson Lars-Ake. Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1-containing endosomes. J Biol Chem. 2002 Sep 10;277(46):44431–44439. doi: 10.1074/jbc.M205241200. [DOI] [PubMed] [Google Scholar]
  3. Chuang W. L., Christ M. D., Peng J., Rabenstein D. L. An NMR and molecular modeling study of the site-specific binding of histamine by heparin, chemically modified heparin, and heparin-derived oligosaccharides. Biochemistry. 2000 Apr 4;39(13):3542–3555. doi: 10.1021/bi9926025. [DOI] [PubMed] [Google Scholar]
  4. David G., Bai X. M., Van der Schueren B., Cassiman J. J., Van den Berghe H. Developmental changes in heparan sulfate expression: in situ detection with mAbs. J Cell Biol. 1992 Nov;119(4):961–975. doi: 10.1083/jcb.119.4.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dempsey L. A., Plummer T. B., Coombes S. L., Platt J. L. Heparanase expression in invasive trophoblasts and acute vascular damage. Glycobiology. 2000 May;10(5):467–475. doi: 10.1093/glycob/10.5.467. [DOI] [PubMed] [Google Scholar]
  6. Dong J., Kukula A. K., Toyoshima M., Nakajima M. Genomic organization and chromosome localization of the newly identified human heparanase gene. Gene. 2000 Aug 8;253(2):171–178. doi: 10.1016/s0378-1119(00)00251-1. [DOI] [PubMed] [Google Scholar]
  7. Egeblad Mikala, Werb Zena. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer. 2002 Mar;2(3):161–174. doi: 10.1038/nrc745. [DOI] [PubMed] [Google Scholar]
  8. Fairbanks M. B., Mildner A. M., Leone J. W., Cavey G. S., Mathews W. R., Drong R. F., Slightom J. L., Bienkowski M. J., Smith C. W., Bannow C. A. Processing of the human heparanase precursor and evidence that the active enzyme is a heterodimer. J Biol Chem. 1999 Oct 15;274(42):29587–29590. doi: 10.1074/jbc.274.42.29587. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Friedmann Y., Vlodavsky I., Aingorn H., Aviv A., Peretz T., Pecker I., Pappo O. Expression of heparanase in normal, dysplastic, and neoplastic human colonic mucosa and stroma. Evidence for its role in colonic tumorigenesis. Am J Pathol. 2000 Oct;157(4):1167–1175. doi: 10.1016/S0002-9440(10)64632-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gallagher J. T., Walker A. Molecular distinctions between heparan sulphate and heparin. Analysis of sulphation patterns indicates that heparan sulphate and heparin are separate families of N-sulphated polysaccharides. Biochem J. 1985 Sep 15;230(3):665–674. doi: 10.1042/bj2300665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Gohji K., Okamoto M., Kitazawa S., Toyoshima M., Dong J., Katsuoka Y., Nakajima M. Heparanase protein and gene expression in bladder cancer. J Urol. 2001 Oct;166(4):1286–1290. [PubMed] [Google Scholar]
  14. Goldshmidt O., Zcharia E., Aingorn H., Guatta-Rangini Z., Atzmon R., Michal I., Pecker I., Mitrani E., Vlodavsky I. Expression pattern and secretion of human and chicken heparanase are determined by their signal peptide sequence. J Biol Chem. 2001 May 31;276(31):29178–29187. doi: 10.1074/jbc.M102462200. [DOI] [PubMed] [Google Scholar]
  15. Goldshmidt Orit, Nadav Liat, Aingorn Helena, Irit Cohen, Feinstein Naomi, Ilan Neta, Zamir Eli, Geiger Benjamin, Vlodavsky Israel, Katz Ben Zion. Human heparanase is localized within lysosomes in a stable form. Exp Cell Res. 2002 Nov 15;281(1):50–62. doi: 10.1006/excr.2002.5651. [DOI] [PubMed] [Google Scholar]
  16. Goldshmidt Orit, Zcharia Eyal, Abramovitch Rinat, Metzger Shula, Aingorn Helena, Friedmann Yael, Schirrmacher Volker, Mitrani Eduardo, Vlodavsky Israel. Cell surface expression and secretion of heparanase markedly promote tumor angiogenesis and metastasis. Proc Natl Acad Sci U S A. 2002 Jul 3;99(15):10031–10036. doi: 10.1073/pnas.152070599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Hulett M. D., Hornby J. R., Ohms S. J., Zuegg J., Freeman C., Gready J. E., Parish C. R. Identification of active-site residues of the pro-metastatic endoglycosidase heparanase. Biochemistry. 2000 Dec 26;39(51):15659–15667. doi: 10.1021/bi002080p. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Ikuta M., Podyma K. A., Maruyama K., Enomoto S., Yanagishita M. Expression of heparanase in oral cancer cell lines and oral cancer tissues. Oral Oncol. 2001 Feb;37(2):177–184. doi: 10.1016/s1368-8375(00)00077-4. [DOI] [PubMed] [Google Scholar]
  21. Iversen P. O., Sorensen D. R., Benestad H. B. Inhibitors of angiogenesis selectively reduce the malignant cell load in rodent models of human myeloid leukemias. Leukemia. 2002 Mar;16(3):376–381. doi: 10.1038/sj.leu.2402376. [DOI] [PubMed] [Google Scholar]
  22. Kim Anthony W., Xu Xiulong, Hollinger Edward F., Gattuso Paolo, Godellas Constantine V., Prinz Richard A. Human heparanase-1 gene expression in pancreatic adenocarcinoma. J Gastrointest Surg. 2002 Mar-Apr;6(2):167–172. doi: 10.1016/s1091-255x(01)00087-7. [DOI] [PubMed] [Google Scholar]
  23. Kjellén L., Lindahl U. Proteoglycans: structures and interactions. Annu Rev Biochem. 1991;60:443–475. doi: 10.1146/annurev.bi.60.070191.002303. [DOI] [PubMed] [Google Scholar]
  24. Koliopanos A., Friess H., Kleeff J., Shi X., Liao Q., Pecker I., Vlodavsky I., Zimmermann A., Büchler M. W. Heparanase expression in primary and metastatic pancreatic cancer. Cancer Res. 2001 Jun 15;61(12):4655–4659. [PubMed] [Google Scholar]
  25. Kuberan Balagurunathan, Lech Miroslaw, Zhang Lijuan, Wu Zhengliang L., Beeler David L., Rosenberg Robert D. Analysis of heparan sulfate oligosaccharides with ion pair-reverse phase capillary high performance liquid chromatography-microelectrospray ionization time-of-flight mass spectrometry. J Am Chem Soc. 2002 Jul 24;124(29):8707–8718. doi: 10.1021/ja0178867. [DOI] [PubMed] [Google Scholar]
  26. Kussie P. H., Hulmes J. D., Ludwig D. L., Patel S., Navarro E. C., Seddon A. P., Giorgio N. A., Bohlen P. Cloning and functional expression of a human heparanase gene. Biochem Biophys Res Commun. 1999 Jul 22;261(1):183–187. doi: 10.1006/bbrc.1999.0962. [DOI] [PubMed] [Google Scholar]
  27. Marchetti D., Nicolson G. L. Human heparanase: a molecular determinant of brain metastasis. Adv Enzyme Regul. 2001;41:343–359. doi: 10.1016/s0065-2571(00)00016-9. [DOI] [PubMed] [Google Scholar]
  28. Maxhimer Justin B., Quiros Roderick M., Stewart Robyn, Dowlatshahi Kambiz, Gattuso Paolo, Fan Ming, Prinz Richard A., Xu Xiulong. Heparanase-1 expression is associated with the metastatic potential of breast cancer. Surgery. 2002 Aug;132(2):326–333. doi: 10.1067/msy.2002.125719. [DOI] [PubMed] [Google Scholar]
  29. Miao Hua-Quan, Navarro Elizabeth, Patel Sheetal, Sargent David, Koo Henry, Wan Hong, Plata Anadellys, Zhou Qinwei, Ludwig Dale, Bohlen Peter. Cloning, expression, and purification of mouse heparanase. Protein Expr Purif. 2002 Dec;26(3):425–431. doi: 10.1016/s1046-5928(02)00558-2. [DOI] [PubMed] [Google Scholar]
  30. Mikami S., Ohashi K., Usui Y., Nemoto T., Katsube K., Yanagishita M., Nakajima M., Nakamura K., Koike M. Loss of syndecan-1 and increased expression of heparanase in invasive esophageal carcinomas. Jpn J Cancer Res. 2001 Oct;92(10):1062–1073. doi: 10.1111/j.1349-7006.2001.tb01061.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nadav Liat, Eldor Amiram, Yacoby-Zeevi Oron, Zamir Eli, Pecker Iris, Ilan Neta, Geiger Benjamin, Vlodavsky Israel, Katz Ben-Zion. Activation, processing and trafficking of extracellular heparanase by primary human fibroblasts. J Cell Sci. 2002 May 15;115(Pt 10):2179–2187. doi: 10.1242/jcs.115.10.2179. [DOI] [PubMed] [Google Scholar]
  32. Nader H. B., Porcionatto M. A., Tersariol I. L., Pinhal M. A., Oliveira F. W., Moraes C. T., Dietrich C. P. Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy. J Biol Chem. 1990 Oct 5;265(28):16807–16813. [PubMed] [Google Scholar]
  33. Okada Yukihiko, Yamada Shuhei, Toyoshima Minako, Dong Jian, Nakajima Motowo, Sugahara Kazuyuki. Structural recognition by recombinant human heparanase that plays critical roles in tumor metastasis. Hierarchical sulfate groups with different effects and the essential target disulfated trisaccharide sequence. J Biol Chem. 2002 Sep 3;277(45):42488–42495. doi: 10.1074/jbc.M206510200. [DOI] [PubMed] [Google Scholar]
  34. Overall Christopher Mark, López-Otín Carlos. Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nat Rev Cancer. 2002 Sep;2(9):657–672. doi: 10.1038/nrc884. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Rohloff J., Zinke J., Schoppmeyer K., Tannapfel A., Witzigmann H., Mössner J., Wittekind C., Caca K. Heparanase expression is a prognostic indicator for postoperative survival in pancreatic adenocarcinoma. Br J Cancer. 2002 Apr 22;86(8):1270–1275. doi: 10.1038/sj.bjc.6600232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schor H., Vaday G. G., Lider O. Modulation of leukocyte behavior by an inflamed extracellular matrix. Dev Immunol. 2000;7(2-4):227–238. doi: 10.1155/2000/51902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tang Weihua, Nakamura Yasushi, Tsujimoto Masahiko, Sato Misako, Wang Xiaojuan, Kurozumi Kazushi, Nakahara Masaaki, Nakao Kazuyasu, Nakamura Misa, Mori Ichiro. Heparanase: a key enzyme in invasion and metastasis of gastric carcinoma. Mod Pathol. 2002 Jun;15(6):593–598. doi: 10.1038/modpathol.3880571. [DOI] [PubMed] [Google Scholar]
  39. Tannock I. F., Rotin D. Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res. 1989 Aug 15;49(16):4373–4384. [PubMed] [Google Scholar]
  40. Tarentino A. L., Plummer T. H., Jr Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Methods Enzymol. 1994;230:44–57. doi: 10.1016/0076-6879(94)30006-2. [DOI] [PubMed] [Google Scholar]
  41. Toyoshima M., Nakajima M. Human heparanase. Purification, characterization, cloning, and expression. J Biol Chem. 1999 Aug 20;274(34):24153–24160. doi: 10.1074/jbc.274.34.24153. [DOI] [PubMed] [Google Scholar]
  42. Tumova S., Hatch B. A., Law D. J., Bame K. J. Basic fibroblast growth factor does not prevent heparan sulphate proteoglycan catabolism in intact cells, but it alters the distribution of the glycosaminoglycan degradation products. Biochem J. 1999 Feb 1;337(Pt 3):471–481. [PMC free article] [PubMed] [Google Scholar]
  43. Uchio Edward M., Linehan W. Marston, Figg William D., Walther McClellan M. A phase I study of intravesical suramin for the treatment of superficial transitional cell carcinoma of the bladder. J Urol. 2003 Jan;169(1):357–360. doi: 10.1016/S0022-5347(05)64126-2. [DOI] [PubMed] [Google Scholar]
  44. Uno F., Fujiwara T., Takata Y., Ohtani S., Katsuda K., Takaoka M., Ohkawa T., Naomoto Y., Nakajima M., Tanaka N. Antisense-mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells. Cancer Res. 2001 Nov 1;61(21):7855–7860. [PubMed] [Google Scholar]
  45. Vaday G. G., Lider O. Extracellular matrix moieties, cytokines, and enzymes: dynamic effects on immune cell behavior and inflammation. J Leukoc Biol. 2000 Feb;67(2):149–159. doi: 10.1002/jlb.67.2.149. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Vlodavsky Israel, Goldshmidt Orit, Zcharia Eyal, Atzmon Ruth, Rangini-Guatta Zehava, Elkin Michael, Peretz Tamar, Friedmann Yael. Mammalian heparanase: involvement in cancer metastasis, angiogenesis and normal development. Semin Cancer Biol. 2002 Apr;12(2):121–129. doi: 10.1006/scbi.2001.0420. [DOI] [PubMed] [Google Scholar]
  48. Yamada S., Sakamoto K., Tsuda H., Yoshida K., Sugahara K., Khoo K. H., Morris H. R., Dell A. Structural studies on the tri- and tetrasaccharides isolated from porcine intestinal heparin and characterization of heparinase/heparitinases using them as substrates. Glycobiology. 1994 Feb;4(1):69–78. doi: 10.1093/glycob/4.1.69. [DOI] [PubMed] [Google Scholar]
  49. Yamada S., Yamane Y., Tsuda H., Yoshida K., Sugahara K. A major common trisulfated hexasaccharide core sequence, hexuronic acid(2-sulfate)-glucosamine(N-sulfate)-iduronic acid-N-acetylglucosamine-glucuronic acid-glucosamine(N-sulfate), isolated from the low sulfated irregular region of porcine intestinal heparin. J Biol Chem. 1998 Jan 23;273(4):1863–1871. doi: 10.1074/jbc.273.4.1863. [DOI] [PubMed] [Google Scholar]
  50. van Kuppevelt T. H., Dennissen M. A., van Venrooij W. J., Hoet R. M., Veerkamp J. H. Generation and application of type-specific anti-heparan sulfate antibodies using phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney. J Biol Chem. 1998 May 22;273(21):12960–12966. doi: 10.1074/jbc.273.21.12960. [DOI] [PubMed] [Google Scholar]

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