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. 1995 Apr;4(4):756–764. doi: 10.1002/pro.5560040415

Identification of the binding and activating sites of the sphingolipid activator protein, saposin C, with glucocerebrosidase.

S Weiler 1, Y Kishimoto 1, J S O'Brien 1, J A Barranger 1, J M Tomich 1
PMCID: PMC2143096  PMID: 7613473

Abstract

Saposin C is a sphingolipid activator protein of 8.5 kDa that activates lysosomal glucocerebrosidase. Previously, we synthesized and characterized a synthetic full-length human saposin C protein that displays 85% of the activity of the native saposin C. In this study we use shorter synthetic peptides derived from the saposin C sequence to map binding and activation sites. By determining the activity and kinetic constant (Kact) values of these peptides, we have identified two functional domains, each comprising a binding site adjacent to or partially overlapping with an activation site. Domains 1 and 2 are located within amino acid positions 6-34 and 41-60, respectively. The activation sites span residues 27-34 and 41-49, whereas binding sites encompass residues 6-27 and 45-60. Peptides containing the sequences of either domain displayed 90% of the activity of the full-length synthetic saposin C. Domain 2, however, bound to glucocerebrosidase by at least an order of magnitude more strongly than domain 1. Binding sites within these domains contain sequences that are excellent candidates for forming amphipathic helical structures. Competition assays demonstrated that the binding of one domain to glucocerebrosidase prevents binding of the other domain, and that saposin A and saposin C bind to the same sites on glucocerebrosidase. A model predicting a saposin C:glucocerebrosidase complex with a stoichiometry of 4:2, respectively, is presented.

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Selected References

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  1. Aerts J. M., Donker-Koopman W. E., Murray G. J., Barranger J. A., Tager J. M., Schram A. W. A procedure for the rapid purification in high yield of human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibodies. Anal Biochem. 1986 May 1;154(2):655–663. doi: 10.1016/0003-2697(86)90043-6. [DOI] [PubMed] [Google Scholar]
  2. Aerts J. M., Donker-Koopman W. E., van Laar C., Brul S., Murray G. J., Wenger D. A., Barranger J. A., Tager J. M., Schram A. W. Relationship between the two immunologically distinguishable forms of glucocerebrosidase in tissue extracts. Eur J Biochem. 1987 Mar 16;163(3):583–589. doi: 10.1111/j.1432-1033.1987.tb10907.x. [DOI] [PubMed] [Google Scholar]
  3. Bahnson A. B., Nimgaonkar M., Fei Y., Boggs S. S., Robbins P. D., Ohashi T., Dunigan J., Li J., Ball E. D., Barranger J. A. Transduction of CD34+ enriched cord blood and Gaucher bone marrow cells by a retroviral vector carrying the glucocerebrosidase gene. Gene Ther. 1994 May;1(3):176–184. [PubMed] [Google Scholar]
  4. Basu A., Glew R. H. Characterization of the phospholipid requirement of a rat liver beta-glucosidase. Biochem J. 1984 Dec 1;224(2):515–524. doi: 10.1042/bj2240515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Basu A., Glew R. H., Daniels L. B., Clark L. S. Activators of spleen glucocerebrosidase from controls and patients with various forms of Gaucher's disease. J Biol Chem. 1984 Feb 10;259(3):1714–1719. [PubMed] [Google Scholar]
  6. Basu A., Prence E., Garrett K., Glew R. H., Ellingson J. S. Comparison of N-acyl phosphatidylethanolamines with different N-acyl groups as activators of glucocerebrosidase in various forms of Gaucher's disease. Arch Biochem Biophys. 1985 Nov 15;243(1):28–34. doi: 10.1016/0003-9861(85)90770-2. [DOI] [PubMed] [Google Scholar]
  7. Christomanou H., Aignesberger A., Linke R. P. Immunochemical characterization of two activator proteins stimulating enzymic sphingomyelin degradation in vitro. Absence of one of them in a human Gaucher disease variant. Biol Chem Hoppe Seyler. 1986 Sep;367(9):879–890. doi: 10.1515/bchm3.1986.367.2.879. [DOI] [PubMed] [Google Scholar]
  8. Christomanou H., Chabás A., Pámpols T., Guardiola A. Activator protein deficient Gaucher's disease. A second patient with the newly identified lipid storage disorder. Klin Wochenschr. 1989 Oct 2;67(19):999–1003. doi: 10.1007/BF01716064. [DOI] [PubMed] [Google Scholar]
  9. Dale G. L., Villacorte D. G., Beutler E. Solubilization of glucocerebrosidase from human placenta and demonstration of a phospholipid requirement for its catalytic activity. Biochem Biophys Res Commun. 1976 Aug 23;71(4):1048–1053. doi: 10.1016/0006-291x(76)90760-9. [DOI] [PubMed] [Google Scholar]
  10. Fabbro D., Grabowski G. A. Human acid beta-glucosidase. Use of inhibitory and activating monoclonal antibodies to investigate the enzyme's catalytic mechanism and saposin A and C binding sites. J Biol Chem. 1991 Aug 15;266(23):15021–15027. [PubMed] [Google Scholar]
  11. Fujibayashi S., Wenger D. A. Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts. J Biol Chem. 1986 Nov 15;261(32):15339–15343. [PubMed] [Google Scholar]
  12. Furbish F. S., Steer C. J., Krett N. L., Barranger J. A. Uptake and distribution of placental glucocerebrosidase in rat hepatic cells and effects of sequential deglycosylation. Biochim Biophys Acta. 1981 Apr 3;673(4):425–434. doi: 10.1016/0304-4165(81)90474-8. [DOI] [PubMed] [Google Scholar]
  13. HO M. W., Rigby M. Glucocerebrosidase: stoichiometry of association between effector and catalytic proteins. Biochim Biophys Acta. 1975 Jul 27;397(1):267–273. doi: 10.1016/0005-2744(75)90199-0. [DOI] [PubMed] [Google Scholar]
  14. Harzer K., Paton B. C., Poulos A., Kustermann-Kuhn B., Roggendorf W., Grisar T., Popp M. Sphingolipid activator protein deficiency in a 16-week-old atypical Gaucher disease patient and his fetal sibling: biochemical signs of combined sphingolipidoses. Eur J Pediatr. 1989 Oct;149(1):31–39. doi: 10.1007/BF02024331. [DOI] [PubMed] [Google Scholar]
  15. Ho M. W., Light N. D. Glucocerebrosidase: reconstitution from macromolecular components depends on acidic phospholipids. Biochem J. 1973 Nov;136(3):821–823. doi: 10.1042/bj1360821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ho M. W., O'Brien J. S. Gaucher's disease: deficiency of 'acid' -glucosidase and reconstitution of enzyme activity in vitro. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2810–2813. doi: 10.1073/pnas.68.11.2810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ho M. W., O'Brien J. S., Radin N. S., Erickson J. S. Glucocerebrosidase: reconstitution of activity from macromolecular components. Biochem J. 1973 Jan;131(1):173–176. doi: 10.1042/bj1310173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morimoto S., Kishimoto Y., Tomich J., Weiler S., Ohashi T., Barranger J. A., Kretz K. A., O'Brien J. S. Interaction of saposins, acidic lipids, and glucosylceramidase. J Biol Chem. 1990 Feb 5;265(4):1933–1937. [PubMed] [Google Scholar]
  19. Nimgaonkar M. T., Bahnson A. B., Boggs S. S., Ball E. D., Barranger J. A. Transduction of mobilized peripheral blood CD34+ cells with the glucocerebrosidase cDNA. Gene Ther. 1994 May;1(3):201–207. [PubMed] [Google Scholar]
  20. O'Brien J. S., Kishimoto Y. Saposin proteins: structure, function, and role in human lysosomal storage disorders. FASEB J. 1991 Mar 1;5(3):301–308. doi: 10.1096/fasebj.5.3.2001789. [DOI] [PubMed] [Google Scholar]
  21. Ohashi T., Boggs S., Robbins P., Bahnson A., Patrene K., Wei F. S., Wei J. F., Li J., Lucht L., Fei Y. Efficient transfer and sustained high expression of the human glucocerebrosidase gene in mice and their functional macrophages following transplantation of bone marrow transduced by a retroviral vector. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11332–11336. doi: 10.1073/pnas.89.23.11332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Paton B. C., Schmid B., Kustermann-Kuhn B., Poulos A., Harzer K. Additional biochemical findings in a patient and fetal sibling with a genetic defect in the sphingolipid activator protein (SAP) precursor, prosaposin. Evidence for a deficiency in SAP-1 and for a normal lysosomal neuraminidase. Biochem J. 1992 Jul 15;285(Pt 2):481–488. doi: 10.1042/bj2850481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Prence E., Chakravorti S., Basu A., Clark L. S., Glew R. H., Chambers J. A. Further studies on the activation of glucocerebrosidase by a heat-stable factor from Gaucher spleen. Arch Biochem Biophys. 1985 Jan;236(1):98–109. doi: 10.1016/0003-9861(85)90609-5. [DOI] [PubMed] [Google Scholar]
  24. Sarin V. K., Kent S. B., Tam J. P., Merrifield R. B. Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal Biochem. 1981 Oct;117(1):147–157. doi: 10.1016/0003-2697(81)90704-1. [DOI] [PubMed] [Google Scholar]
  25. Schnabel D., Schröder M., Fürst W., Klein A., Hurwitz R., Zenk T., Weber J., Harzer K., Paton B. C., Poulos A. Simultaneous deficiency of sphingolipid activator proteins 1 and 2 is caused by a mutation in the initiation codon of their common gene. J Biol Chem. 1992 Feb 15;267(5):3312–3315. [PubMed] [Google Scholar]
  26. Sheh L., Glew R. H., Bothner-By A. A., Mishra P. K. High-resolution proton nuclear magnetic resonance studies of the glucocerebrosidase activator protein from Gaucher spleen. Biochemistry. 1985 Nov 5;24(23):6645–6651. doi: 10.1021/bi00344a052. [DOI] [PubMed] [Google Scholar]
  27. Stevens R. L., Faull K. F., Conklin K. A., Green B. N., Fluharty A. L. Porcine cerebroside sulfate activator: further structural characterization and disulfide identification. Biochemistry. 1993 Apr 20;32(15):4051–4059. doi: 10.1021/bi00066a028. [DOI] [PubMed] [Google Scholar]

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