Advertisement

The Sulfs: Expression, Purification, and Substrate Specificity

  • Kenji UchimuraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1229)

Abstract

Sulf-1 and Sulf-2 are endo-acting extracellular sulfatases. The Sulfs liberate 6-O sulfate groups, mainly from N, 6-O, and 2-O trisulfated disaccharides of heparan sulfate (HS)/heparin chains. The Sulfs have been shown to modulate the interaction of a number of protein ligands including growth factors and morphogens with HS/heparin and thus regulate the signaling of these ligands. They also play important roles in development and are dysregulated in many cancers. The establishment of the expression of the Sulfs and methods of assaying them has been desirable to investigate these enzymes. In this chapter, methods to express and purify recombinant Sulfs and to analyze HS structures in an extracellular fraction of HSulf-transfected HEK293 cells are described. The application of these enzymes for ex vivo degradation of an anti-HS epitope accumulated in the brain of a neurodegenerative disease model mouse is also described.

Key words

Heparan sulfate Sulf-1 Sulf-2 Sulfatase Endo-acting enzyme Protein ligands Immunoblot Transfection FLAG-tag His-tag 

Notes

Acknowledgements

Supported by Japanese Health and Labour Sciences Research Grants [H19-001 and H22-007], Grants-in-Aid from the Ministry of Education, Science, Sports and Culture [22790303 and 24590349, and Scientific Research on Innovative Areas] and in part by the Takeda Science Foundation.

References

  1. 1.
    Diez-Roux G, Ballabio A (2005) Sulfatases and human disease. Annu Rev Genomics Hum Genet 6:355–379PubMedCrossRefGoogle Scholar
  2. 2.
    Dhoot GK, Gustafsson MK, Ai X et al (2001) Regulation of Wnt signaling and embryo patterning by an extracellular sulfatase. Science 293:1663–1666PubMedCrossRefGoogle Scholar
  3. 3.
    Morimoto-Tomita M, Uchimura K, Werb Z et al (2002) Cloning and characterization of two extracellular heparin-degrading endosulfatases in mice and humans. J Biol Chem 277:49175–49185PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Ai X, Do AT, Kusche-Gullberg M et al (2006) Substrate specificity and domain functions of extracellular heparan sulfate 6-O-endosulfatases, QSulf1 and QSulf2. J Biol Chem 281:4969–4976PubMedCrossRefGoogle Scholar
  5. 5.
    Frese MA, Milz F, Dick M et al (2009) Characterization of the human sulfatase Sulf1 and its high affinity heparin/heparan sulfate interaction domain. J Biol Chem 284:28033–28044PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Ohto T, Uchida H, Yamazaki H et al (2002) Identification of a novel nonlysosomal sulphatase expressed in the floor plate, choroid plexus and cartilage. Genes Cells 7:173–185PubMedCrossRefGoogle Scholar
  7. 7.
    Nagamine S, Koike S, Keino-Masu K et al (2005) Expression of a heparan sulfate remodeling enzyme, heparan sulfate 6-O-endosulfatase sulfatase FP2, in the rat nervous system. Brain Res Dev Brain Res 159:135–143PubMedCrossRefGoogle Scholar
  8. 8.
    Rosen SD, Lemjabbar-Alaoui H (2010) Sulf-2: an extracellular modulator of cell signaling and a cancer target candidate. Expert Opin Ther Targets 14:935–949PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Dierks T, Schmidt B, Borissenko LV et al (2003) Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme. Cell 113:435–444PubMedCrossRefGoogle Scholar
  10. 10.
    Cosma MP, Pepe S, Annunziata I et al (2003) The multiple sulfatase deficiency gene encodes an essential and limiting factor for the activity of sulfatases. Cell 113:445–456PubMedCrossRefGoogle Scholar
  11. 11.
    Ambasta RK, Ai X, Emerson CP Jr (2007) Quail Sulf1 function requires asparagine-linked glycosylation. J Biol Chem 282:34492–34499PubMedCrossRefGoogle Scholar
  12. 12.
    Tang R, Rosen SD (2009) Functional consequences of the subdomain organization of the sulfs. J Biol Chem 284:21505–21514PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Nagamine S, Keino-Masu K, Shiomi K et al (2010) Proteolytic cleavage of the rat heparan sulfate 6-O-endosulfatase SulfFP2 by furin-type proprotein convertases. Biochem Biophys Res Commun 391:107–112PubMedCrossRefGoogle Scholar
  14. 14.
    Saad OM, Ebel H, Uchimura K et al (2005) Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase. Glycobiology 15:818–826PubMedCrossRefGoogle Scholar
  15. 15.
    Ai X, Do AT, Lozynska O et al (2003) QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling. J Cell Biol 162:341–351PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Viviano BL, Paine-Saunders S, Gasiunas N et al (2004) Domain-specific modification of heparan sulfate by Qsulf1 modulates the binding of the bone morphogenetic protein antagonist Noggin. J Biol Chem 279:5604–5611PubMedCrossRefGoogle Scholar
  17. 17.
    Hossain MM, Hosono-Fukao T, Tang R et al (2010) Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88. Glycobiology 20:175–186PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Dai Y, Yang Y, MacLeod V et al (2005) HSulf-1 and HSulf-2 are potent inhibitors of myeloma tumor growth in vivo. J Biol Chem 280:40066–40073PubMedCrossRefGoogle Scholar
  19. 19.
    Lamanna WC, Baldwin RJ, Padva M et al (2006) Heparan sulfate 6-O-endosulfatases: discrete in vivo activities and functional co-operativity. Biochem J 400:63–73PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Lamanna WC, Frese MA, Balleininger M et al (2008) Sulf loss influences N-, 2-O-, and 6-O-sulfation of multiple heparan sulfate proteoglycans and modulates fibroblast growth factor signaling. J Biol Chem 283:27724–27735PubMedCrossRefGoogle Scholar
  21. 21.
    Nagamine S, Tamba M, Ishimine H et al (2012) Organ-specific sulfation patterns of heparan sulfate generated by extracellular sulfatases Sulf1 and Sulf2 in mice. J Biol Chem 287:9579–9590PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Bishop JR, Schuksz M, Esko JD (2007) Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446:1030–1037PubMedCrossRefGoogle Scholar
  23. 23.
    Bernfield M, Gotte M, Park PW et al (1999) Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68:729–777PubMedCrossRefGoogle Scholar
  24. 24.
    Gallagher JT (2001) Heparan sulfate: growth control with a restricted sequence menu. J Clin Invest 108:357–361PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Nakato H, Kimata K (2002) Heparan sulfate fine structure and specificity of proteoglycan functions. Biochim Biophys Acta 1573:312–318PubMedCrossRefGoogle Scholar
  26. 26.
    Nawroth R, van Zante A, Cervantes S et al (2007) Extracellular sulfatases, elements of the Wnt signaling pathway, positively regulate growth and tumorigenicity of human pancreatic cancer cells. PLoS One 2:e392PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Lemjabbar-Alaoui H, van Zante A, Singer MS et al (2010) Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis. Oncogene 29:635–646PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Phillips JJ, Huillard E, Robinson AE et al (2012) Heparan sulfate sulfatase SULF2 regulates PDGFRalpha signaling and growth in human and mouse malignant glioma. J Clin Invest 122:911–922PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Otsuki S, Hanson SR, Miyaki S et al (2010) Extracellular sulfatases support cartilage homeostasis by regulating BMP and FGF signaling pathways. Proc Natl Acad Sci U S A 107:10202–10207PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Lai J, Chien J, Staub J et al (2003) Loss of HSulf-1 up-regulates heparin-binding growth factor signaling in cancer. J Biol Chem 278:23107–23117PubMedCrossRefGoogle Scholar
  31. 31.
    Wang S, Ai X, Freeman SD et al (2004) QSulf1, a heparan sulfate 6-O-endosulfatase, inhibits fibroblast growth factor signaling in mesoderm induction and angiogenesis. Proc Natl Acad Sci U S A 101:4833–4838PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Li J, Kleeff J, Abiatari I et al (2005) Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer. Mol Cancer 4:14PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Narita K, Staub J, Chien J et al (2006) HSulf-1 inhibits angiogenesis and tumorigenesis in vivo. Cancer Res 66:6025–6032PubMedCrossRefGoogle Scholar
  34. 34.
    Lai JP, Chien JR, Moser DR et al (2004) hSulf1 Sulfatase promotes apoptosis of hepatocellular cancer cells by decreasing heparin-binding growth factor signaling. Gastroenterology 126:231–248PubMedCrossRefGoogle Scholar
  35. 35.
    Lai JP, Chien J, Strome SE et al (2004) HSulf-1 modulates HGF-mediated tumor cell invasion and signaling in head and neck squamous carcinoma. Oncogene 23:1439–1447PubMedCrossRefGoogle Scholar
  36. 36.
    Yue X, Li X, Nguyen HT et al (2008) Transforming growth factor-beta1 induces heparan sulfate 6-O-endosulfatase 1 expression in vitro and in vivo. J Biol Chem 283:20397–20407PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Ai X, Kitazawa T, Do AT et al (2007) SULF1 and SULF2 regulate heparan sulfate-mediated GDNF signaling for esophageal innervation. Development 134:3327–3338PubMedCrossRefGoogle Scholar
  38. 38.
    Lai JP, Sandhu DS, Yu C et al (2008) Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma. Hepatology 47:1211–1222PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Uchimura K, Morimoto-Tomita M, Bistrup A et al (2006) HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1. BMC Biochem 7:2PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Morimoto-Tomita M, Uchimura K, Bistrup A et al (2005) Sulf-2, a proangiogenic heparan sulfate endosulfatase, is upregulated in breast cancer. Neoplasia 7:1001–1010PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Fujita K, Takechi E, Sakamoto N et al (2010) HpSulf, a heparan sulfate 6-O-endosulfatase, is involved in the regulation of VEGF signaling during sea urchin development. Mech Dev 127:235–245PubMedCrossRefGoogle Scholar
  42. 42.
    Freeman SD, Moore WM, Guiral EC et al (2008) Extracellular regulation of developmental cell signaling by XtSulf1. Dev Biol 320:436–445PubMedCrossRefGoogle Scholar
  43. 43.
    Ratzka A, Kalus I, Moser M et al (2008) Redundant function of the heparan sulfate 6-O-endosulfatases Sulf1 and Sulf2 during skeletal development. Dev Dyn 237:339–353PubMedCrossRefGoogle Scholar
  44. 44.
    Lum DH, Tan J, Rosen SD et al (2007) Gene trap disruption of the mouse heparan sulfate 6-O-endosulfatase gene, Sulf2. Mol Cell Biol 27:678–688PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Holst CR, Bou-Reslan H, Gore BB et al (2007) Secreted sulfatases Sulf1 and Sulf2 have overlapping yet essential roles in mouse neonatal survival. PLoS One 2:e575PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Kalus I, Salmen B, Viebahn C et al (2009) Differential involvement of the extracellular 6-O-endosulfatases Sulf1 and Sulf2 in brain development and neuronal and behavioral plasticity. J Cell Mol Med 13:4505–4521PubMedCrossRefGoogle Scholar
  47. 47.
    Maltseva I, Chan M, Kalus I et al (2013) The SULFs, extracellular sulfatases for heparan sulfate, promote the migration of corneal epithelial cells during wound repair. PLoS One 8:e69642PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Toyoda H, Kinoshita-Toyoda A, Fox B et al (2000) Structural analysis of glycosaminoglycans in animals bearing mutations in sugarless, sulfateless, and tout-velu. Drosophila homologues of vertebrate genes encoding glycosaminoglycan biosynthetic enzymes. J Biol Chem 275:21856–21861PubMedCrossRefGoogle Scholar
  49. 49.
    Hosono-Fukao T, Ohtake-Niimi S, Nishitsuji K et al (2011) RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature. J Neurosci Res 89:1840–1848PubMedCrossRefGoogle Scholar
  50. 50.
    Jenniskens GJ, Oosterhof A, Brandwijk R et al (2000) Heparan sulfate heterogeneity in skeletal muscle basal lamina: demonstration by phage display-derived antibodies. J Neurosci 20:4099–4111PubMedGoogle Scholar
  51. 51.
    Dennissen MA, Jenniskens GJ, Pieffers M et al (2002) Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies. J Biol Chem 277:10982–10986PubMedCrossRefGoogle Scholar
  52. 52.
    Jenniskens GJ, Hafmans T, Veerkamp JH et al (2002) Spatiotemporal distribution of heparan sulfate epitopes during myogenesis and synaptogenesis: a study in developing mouse intercostal muscle. Dev Dyn 225:70–79PubMedCrossRefGoogle Scholar
  53. 53.
    Uchimura K, Lemjabbar-Alaoui H, van Kuppevelt TH et al (2010) Use of a phage display antibody to measure the enzymatic activity of the Sulfs. Methods Enzymol 480:51–64PubMedCrossRefGoogle Scholar
  54. 54.
    Hosono-Fukao T, Ohtake-Niimi S, Hoshino H et al (2012) Heparan sulfate subdomains that are degraded by Sulf accumulate in cerebral amyloid ss plaques of Alzheimer’s disease: evidence from mouse models and patients. Am J Pathol 180:2056–2067PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of BiochemistryNagoya University Graduate School of MedicineNagoyaJapan

Personalised recommendations