Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structure of human β-glucuronidase reveals candidate lysosomal targeting and active-site motifs

Nature Structural Biology volume 3, pages 375–381 (1996)Cite this article

Abstract

The X-ray structure of the homotetrameric lysosomal acid hydrolase, human β-glucuronidase (332,000 Mr), has been determined at 2.6 Å resolution. The tetramer has approximate dihedral symmetry and each protomer consists of three structural domains with topologies similar to a jelly roll barrel, an immunoglobulin constant domain and a TIM barrel respectively. Residues 179–204 form a β-hairpin motif similar to the putative lysosomal targeting motif of cathepsin D, supporting the view that lysosomal targeting has a structural basis. The active site of the enzyme is formed from a large cleft at the interface of two monomers. Residues Glu 451 and Glu 540 are proposed to be important for catalysis. The structure establishes a framework for understanding mutations that lead to the human genetic disease mucopolysaccharidosis VII, and for using the enzyme in anti-cancer therapy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Paigen, K. Mammalian β-Glucuronidase: Genetics, molecular biology and cell biology. Progr. Nucleic Acid Res. Molec. Biol. 37, 155–205 (1989).

    Article  CAS  Google Scholar 

  2. Oshima, W. et al. Cloning, sequencing, and expression of cDNA for human β-Glucuronidase. Proc. Natl. Acad. Sci. USA 84, 685–689 (1987).

    Article  CAS  Google Scholar 

  3. Shipley, J.M., Grubb, J.H. & Sly, W.S. The role of glycosylation and phosphorylation in the expression of active human β-Glucuronidase. J. Biol. Chem. 268, 12193–12198 (1993).

    CAS  PubMed  Google Scholar 

  4. Kaplan, A., Achord, D.T. & Sly, W.S. Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts. Proc. Natl. Acad. Sci. USA 74, 2026–2030 (1977).

    Article  CAS  Google Scholar 

  5. Natowicz, M.R., Chi, M.M.-Y., Lowry, O.H. & Sly, W.S. Enzymatic identification of mannose-6-phosphate on the recognition marker for receptor-mediated pinocytosis of β-Glucuronidase by human fibroblasts. Proc Natl. Acad. Sci. USA 76, 4322–4326 (1979).

    Article  CAS  Google Scholar 

  6. Dahms, N.M., Lobel, P. & Kornfeld, S. Mannose 6-phosphate receptors and lysosomal enzyme targeting. J. Biol. Chem. 264, 12115–12118 (1989).

    CAS  PubMed  Google Scholar 

  7. Reitman, M.L. & Kornfeld, S. UDP-Nacetylglucosamine: Glycoprotein N- acetylglucosamine-1-phosphotransferase, proposed enzyme for the phosphorylation of the high mannose oligosaccharide units of lysosomal enzymes. J. Biol. Chem. 256, 4275–4281 (1981).

    CAS  PubMed  Google Scholar 

  8. Waheed, A., Hasilik, A. & von Figura, K. Processing of the phosphorylated recognition marker in lysosomal enzymes. J. Biol. Chem. 256, 5717–5721 (1981).

    CAS  PubMed  Google Scholar 

  9. Lang, L., Reitman, M.L., Tang, J., Roberts, R.M. & Kornfeld, S. Recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes. J. Biol. Chem. 259, 14663–14671 (1984).

    CAS  PubMed  Google Scholar 

  10. Baranski, T.J., Faust, P.L. & Kornfeld, S. Generation of a lysosomal enzyme targeting signal in the secretory protein pepsinogen. Cell 63, 281–291 (1990).

    Article  CAS  Google Scholar 

  11. Baranski, T.J., Koelsch, G., Hartsuck, J.A. & Kornfeld, S. Mapping and molecular modeling of a recognition domain for lysosomal enzyme targeting. J. Biol. Chem. 266, 23365–23372 (1991).

    CAS  PubMed  Google Scholar 

  12. Metcalf, P. & Fusek, M. Two crystal structure for cathepsin D: The lysosomal targeting signal and active site. EMBO J. 12, 1293–1302 (1993).

    Article  CAS  Google Scholar 

  13. Musil, D. et al. The refined 2.15 Å x-ray crystal structure of human liver cathepsin B: The structural basis for its specificity. EMBO J. 10, 2321–2330 (1991).

    Article  CAS  Google Scholar 

  14. Jia, Z. et al. Crystal structure of recombinant rat cathepsin B and a cathepsin B-inhibitor complex. Implications for structure-based inhibitor design. J. Biol. Chem. 270, 5527–5533 (1995)

    Article  CAS  Google Scholar 

  15. Lin, X., Dashti, A., Schinazi, R.F. & Tang, J. Intracellular diversion of glycoprotein GP160 of human immunodeficiency virus to lysosomes as a strategy of AIDS gene therapy. FASEB J. 7, 1070–1080 (1993).

    Article  CAS  Google Scholar 

  16. Bosslet, K. et al. Molecular and functional characterization of a fusion protein suited for tumor specific prodrug activation. Br. J. Cancer 65, 234–238 (1992).

    Article  CAS  Google Scholar 

  17. Bosslet, K., Czech, J. & Hoffman, D. Tumor-selective prodrug activation by fusion protein-mediated catalysis. Cancer Res. 54, 2151–2159 (1994).

    CAS  PubMed  Google Scholar 

  18. Caygill, J.C. & Pitkeathy, D.A. A Study of β-Acetylglucosaminase and acid phosphatase in pathological joint fluids. Ann. Rheum. Dis. 25, 137–145 (1966).

    Article  CAS  Google Scholar 

  19. Weissman, G., Zurier, R.B. & Spieler, P.Z. Mechanisms of lysosomal enzyme release from leukocytes exposed to immune complexes and other particles. J. Exp. Med. 134, 521–536 (1971).

    Google Scholar 

  20. Boyer, M.J. & Tannock, I.F. Lysosomes, lysosomal enzymes, and cancer. Adv. Cancer Res. 60, 269–291 (1993).

    Article  CAS  Google Scholar 

  21. Kinoshita, N. & Gelboin, H.V. β-Glucuronidase catalyzed hydrolysis of benzo[a]pyrene-3-glucuronide and binding to DNA. Science 199, 307–309 (1978).

    Article  CAS  Google Scholar 

  22. Kim, D.-H., Kang, H.-J., Park, S.-H. & Kobashi, K. Characterization of β- glucosidase and β-Glucuronidase of alkalotolerant intestinal bacteria. Biol. Pharm. Bull. 17, 423–426 (1994).

    Article  CAS  Google Scholar 

  23. Sly, W.S., Quinton, B.A., McAlister, W.H. & Rimoin, D.L. Beta-glucuronidase deficiency: Report of clinical, radiologic, and biochemical features of a new mucopolysaccharidosis. J. Pediatr. 82, 249–257 (1973).

    Article  CAS  Google Scholar 

  24. Tomatsu, S. et al. Mucopolysaccharidosis type VII: Characterization of mutations and molecular heterogeneity. Am. J. Hum. Genet. 48, 89–96 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Shipley, J.M. et al. Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes. Am. J. Hum. Genet 52, 517–526 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Wu, B.M., Tomatsu, S., Sukegawa, K., Orii, T. & Sly, W.S. Overexpression rescues the mutant phenotype of I176f mutation causing β-Glucuronidase deficiency mucopolysaccharidosis in two Mennonite siblings. J. Biol. Chem. 269, 23681–23688 (1994).

    CAS  PubMed  Google Scholar 

  27. Vervoort, R., Lissens, W. & Liebaers, I. Molecular analysis of a patient with hydrops fatalis caused by β-Glucuronidase deficiency, and evidence for additional pseudogenes. Hum. Mutat. 2, 443–445 (1993).

    Article  CAS  Google Scholar 

  28. Drendel, W.B. et al. Crystallization and Preliminary Crystallographic Studies of Human β-Glucuronidase. J. Mol. Biol. 233, 173–176 (1993).

    Article  CAS  Google Scholar 

  29. Brünger, A.T. The free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–474 (1992).

    Article  Google Scholar 

  30. Hardman, K.D. & Ainsworth, C.F. Structure of concavalin A at 2.4 Å resolution. Biochem. 11, 4910–4919 (1972).

    Article  CAS  Google Scholar 

  31. Alzari, P.N., Lascombe, M.-B. & Poljak, R.J. Three dimensional structure of antibodies. Annu. Rev. Immunol. 6, 555–580 (1988).

    Article  CAS  Google Scholar 

  32. Muirhead, H. Triose phosphate isomerase, pyruvate kinase and other α/β-barrel enzymes. Trends Biochem. Sci. 8, 326–330 (1983).

    Article  CAS  Google Scholar 

  33. Jacobson, R.H., Zhang, X.-J., DuBose, R.F. & Matthews, B.W. Three-dimensional structure of β-galactosidase from E. Coli. Nature 369, 761–766 (1994).

    Article  CAS  Google Scholar 

  34. Faust, P. Lysosomal enzyme phosphorylation: analysis of the lysosomal enzyme protein recognition domain in the aspartyl protease gene family. Ph.D. Dissertation, Washington University School of Medicine, St. Louis, MO (1988).

  35. Cuozzo, J.W., Tao, K., Wu, Q., Young, W. & Sahagian, G.G. Lysine-based structure in the proregion of procathepsin I is the recognition site for mannose phosphorylation. J. Biol. Chem. 270, 15611–15619 (1995).

    Article  CAS  Google Scholar 

  36. Schorey, J.S., Fortenberry, S.C. & Chirgwin, J.M. Lysine residues in the c- terminal lobe and lysosomal targeting of procathepsin D. J. Cell Sci. 108, 2007–2015 (1995).

    CAS  PubMed  Google Scholar 

  37. Wang, C.-C. & Touster, O. Studies of catalysis by β-Glucuronidase. J. Biol. Chem. 247, 2644–2656 (1972).

    CAS  PubMed  Google Scholar 

  38. Ring, M. & Huber, R.E. Multiple replacements establish the Importance of tyrosine-503 in β-galactosidase (Escherichia coli). Arch. Biochem. Biophys. 283, 342–350 (1990).

    Article  CAS  Google Scholar 

  39. Cupples, C.G., Miller, J.H. & Huber, R.E. Determination of the roles of Glu-461 in β- galactosidase (Escherichia coli) using site-specific mutagenesis. J. Biol. Chem. 265, 5512– 5518 (1990).

    CAS  PubMed  Google Scholar 

  40. Gebler, J.C., Aebersold, R. & Withers, S.G. Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) β-galactosidase from Escherichia coli. J. Biol. Chem. 267, 11126–11130 (1992).

    CAS  PubMed  Google Scholar 

  41. Phillips, D.C. The three dimensional structure of an enzyme molecule. Sci. Amer. 215 (5), 78–90 (1966).

    Article  CAS  Google Scholar 

  42. Jefferson, R.A., Burgess, S.M. & Hirsh, D. β-Glucuronidase from Escherichia coil as a gene-fusion marker. Proc. Natl. Acad. Sci. USA 83, 8447–8451 (1986).

    Article  CAS  Google Scholar 

  43. Russel, A.J. & Fersht, A.R. Rational modification of enzyme catalysis by engineering surface charge. Nature 328, 496–500 (1987).

    Article  Google Scholar 

  44. Gehrmann, M.C., Opper, M., Sedlacek, H.H., Bosslet, K. & Czech, J. Biochemical properties of recombinant human β-Glucuronidase synthesized in baby hamster kidney cells. Biochem. J. 301, 821–828 (1994).

    Article  CAS  Google Scholar 

  45. Watenpaugh, D.D. Overview of phasing by isomorphous replacement. Methods Enzymol. 115, 3–15 (1985).

    Article  CAS  Google Scholar 

  46. Lebioda, L. & Zhang, E. Soaking of crystals for macromolecular crystallography in a capillary. J. Appl. Crystallogr. 25, 323–324 (1992).

    Article  Google Scholar 

  47. Zhang, K.Y.J. SQUASH - Combining constrains for macromolecular phase refinement and extension. Acta Crystallogr. D49, 213–222 (1993).

    CAS  Google Scholar 

  48. Islam, M.R., Grubb, J.H. & Sly, W.S. C-terminal processing of human β-Glucuronidase: The propeptide is required for full expression of catalytic activity, intracellular retention, and proper phosphorylation. J. Biol. Chem. 268, 22627–22632 (1993).

    CAS  PubMed  Google Scholar 

  49. Sack, J.S. CHAIN - a crystallographic modeling program. J. Mol. Graphics 6, 224–235 (1988).

    Article  Google Scholar 

  50. Brunger, A. X-PLOR version 3.1, Yale University Press, New Haven, CT (1992).

    Google Scholar 

  51. Engh, R.A. & Huber, R. Accurate bond and angle parameters for x-ray protein structure refinement. Acta Crystallogr. A47, 392–400 (1991).

    Article  CAS  Google Scholar 

  52. Devereux, J., Haeberli, P. & Smithies, O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research. 12, 387 (1984).

    Article  CAS  Google Scholar 

  53. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

Download references

Author information

Author notes

  1. Sanjeev Jain

    Present address: Department of Internal Medicine, University of Texas Health Sciences Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas, 78284, USA

Authors and Affiliations

  1. E.A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, 63104, USA

    Sanjeev Jain, William B. Drendel, William S. Sly & Jeffrey H. Grubb

  2. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, 63110, USA

    Zhi-wei Chen & F. Scott Mathews

Authors
  1. Sanjeev Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William B. Drendel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhi-wei Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. Scott Mathews
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William S. Sly
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeffrey H. Grubb
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jain, S., Drendel, W., Chen, Zw. et al. Structure of human β-glucuronidase reveals candidate lysosomal targeting and active-site motifs. Nat Struct Mol Biol 3, 375–381 (1996). https://doi.org/10.1038/nsb0496-375

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb0496-375

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing