Studies of SAP-1 and SAP-2 in Cultured Skin Fibroblasts

  • David A. Wenger
  • Shinsuke Fujibayashi
Part of the NATO ASI Series book series (NSSA, volume 116)


Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that activate the hydrolysis of sphingolipids in the presence of the specific lysosomal hydrolases.l The exact number of such proteins and their exact mechanisms of action are not known at this time. However, three proteins with the ability to stimulate the enzymatic hydrolysis of certain sphingolipids have been identified. By our nomenclature SAP-1 has been demonstrated to activate the hydrolysis of sulfatide, GM1 ganglioside and, possibly, globotriaosylceramide by arylsulfatase A, acid β ß-galactosidase and α-galactodidase A, respectively.2–6 SAP-1 is missing in tissue and urine samples from patients with a variant form of metachromatic leukodystrophy.7–10 SAP-2 (factor P, heat-stable factor, cohydrolase SPHI) activates the enzymatic hydrolysis of glucosylceramide (glc-cer), galactosylceramide (gal-cer) and, possibly, sphingomyelin by glucocerebrosi-dase, galactocerebrosidase and sphingomyelinase, respectively.11–15 SAP-2 appears to be normal in tissue samples and fibroblast extracts from patients with Gaucher disease, Krabbe disease and Niemann-Pick disease (including Type C).16 SAP-3 (GM2 activator protein) stimulates the enzymatic hydrolysis of GM2 ganglioside by hexosaminidase A. 17,18 It is deficient in tissue samples and cultured cells from patients with the AB variant form of GM2 gangliosidosis.18–20 There has been some discrepancy about the sizes of native and denatured SAP. In order to learn more about the structure of SAP-1 and SAP-2, their mechanism of action and the nature of the mutations in patients with SAP-1 deficiency the structure and biosynthesis of these SAP were studied in cultured human skin fibroblasts. Using specific antibodies to immunoprecipitate the SAP and to detect the immuno-reactive bands after electrophoresis, some interesting features of these proteins were obtained. These may help us to understand how these proteins regulate sphingolipid metabolism.


Gauche Disease Lysosomal Storage Disease Metachromatic Leukodystrophy High Molecular Weight Species High Molecular Weight Form 
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  1. 1.
    D.A. Wenger and K. Inui, Studies on the sphingolipid activator protein for the enzymatic hydrolysis of GM1 ganglioside and sulfatide, in “Molecular Basis of Lysosomal Storage Disorders,” J.A. Barranger and R.O. Brady, eds., Academic Press, New York (1984).Google Scholar
  2. 2.
    E. Mehl and H. Jatzkewitz, Ein Cerebrosidsulfatase aus schweineniere, Hoppe-Seyler’s Z. Physiol. Chem. 339: 260 (1964).CrossRefGoogle Scholar
  3. 3.
    S.-C. Li and Y.-T. Li, An activator stimulating the enzymic hydrolysis of sphingoglycolipids, J. Biol. Chem. 251: 1159 (1976).PubMedGoogle Scholar
  4. 4.
    K. Inui and D.A. Wenger, Properties of a protein activator of glyco- sphingolipid hydrolysis isolated from the liver of a patient with GM1 gangliosidosis, Type 1, Biochem. Biophys. Res. Commun. 105: 745 (1982).CrossRefGoogle Scholar
  5. 5.
    S. Gartner, E. Conzelmann, and K. Sandhoff, Activator protein for the degradation of globotriaosylceramide by human a-galactosidase, J. Biol. Chem. 258: 12378 (1983).PubMedGoogle Scholar
  6. 6.
    S.-C. Li, H. Kihara, S. Serizawa, Y.-T. Li, A.L. Fluharty, J.S. Mayes, and L.J. Shapiro, Activator protein required for the enzymatic hydrolysis of cerebroside sulfate, J. Biol. Chem. 260: 1867 (1985).PubMedGoogle Scholar
  7. 7.
    L.J. Shapiro, K.A. Aleck, M.M. Kaback, H. Itabashi, R.J. Desnick, N. Brand, R.L. Stevens, A.L. Fluharty, and H. Kihara, Metachromatic leukodystrophy without arylsulfatase A deficiency, Pediatr. Res. 13: 1179 (1979).Google Scholar
  8. 8.
    A.F. Hahn, B.A. Gordon, V. Feleki, G.G. Hinton and J.J. Gilbert, A variant form of metachromatic leukodystrophy without arylsulfatase deficiency, Ann. Neurol. 12: 33 (1982).PubMedCrossRefGoogle Scholar
  9. 9.
    R.L. Stevens, A.L. Fluharty, H. Kihara, M.M. Kaback, L.J. Shapiro, B. Marsh, K. Sandhoff, and G. Fischer, Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy, Am. J. Hum. Genet. 33: 900 (1981).PubMedGoogle Scholar
  10. 10.
    K. Inui, M. Emmett, and D.A. Wenger, Immunological evidence for deficiency in an activator protein for sulfatide sulfatase in a variant form of metachromatic leukodystrophy, Proc. Natl. Acad. Sci. (USA) 80: 3074 (1983).CrossRefGoogle Scholar
  11. 11.
    M.W. Ho and J.S. O’Brien, Gaucher’s disease: deficiency of ‘acid’ ßglucosidase and reconstitution of enzyme activity in vitro, Proc. Natl. Acad. Sci. (USA) 68: 2810 (1971).CrossRefGoogle Scholar
  12. 12.
    S.P. Peters, P. Coyle, C. Coffee, and R.H. Clew, Purification and properties of a heat-stable glucocerebrosidase activating factor from control and Gaucher spleen, J. Biol. Chem. 252: 563 (1977).PubMedGoogle Scholar
  13. 13.
    S.L. Berent and N.S. Radin, ß-glucodidase activator protein from bovine spleen (’co-glucosidase’), Arch. Biochem. Biophys. 208: 248 (1981).Google Scholar
  14. 14.
    D.A. Wenger, M. Sattler, and S. Roth, A protein activator of galactosylceramide ß-galactosidase. Biochim. Biophys. Acta 712: 639 (1982).PubMedCrossRefGoogle Scholar
  15. 15.
    S.S. Iyer, S.L. Berent, and N.S. Radin, The cohydrolases in human spleen that stimulate glucosylceramide Q-glucosidase, Biochim. Biophys. Acta 748: 1 (1983).CrossRefGoogle Scholar
  16. 16.
    S. Fujibayashi and D.A. Wenger, Studies on a sphingolipid activator protein (SAP-2) in fibroblasts from patients with lysosomal storage diseases, including Niemann-Pick disease, Type C, Clin. Chin. Acta 146: 147 (1985).Google Scholar
  17. 17.
    P. Hechtman, Characterization of an activating factor required for hydrolysis of GM2 ganglioside catalyzed by hexosaminidase A, Can. J. Biochem. 55: 315 (1977).Google Scholar
  18. 18.
    E. Conzelmann and K. Sandhoff, AB variant of infantile GM2 gangliosidosis. Deficiency of a factor necessary for stimulation of hexosaminidase A-cataylzed degradation of ganglioside GM2 and glycolipid GA2, Proc. Natl. Acad. Sci. (USA) 75: 3979 (1978).CrossRefGoogle Scholar
  19. 19.
    P. Hechtman, B.A. Gordon, and N.M.K. Ng Ying Kin, Deficiency of the hexosaminidase A activator protein in a case of GM2 gangliosidosis variant AB, Pediatr. Res. 16: 217 (1982).Google Scholar
  20. 20.
    A. Banerjee, J. Burg, E. Conzelmann, M. Carroll, and K. Sandhoff, Enzyme-linked immunosorbent assay for the ganglioside GM2-activator protein, Hoppe-Seyler’s Z. Physiol. Chem. 365: 347 (1984).CrossRefGoogle Scholar
  21. 21.
    U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London) 227: 680 (1970).CrossRefGoogle Scholar
  22. 22.
    W.M. Bonner and R.A. Laskey, A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels, Eur. J. Biochem. 46: 83 (1974).PubMedCrossRefGoogle Scholar
  23. 23.
    K. Inui and D.A. Wenger, Biochemical, immunological, and structural studies on a sphingolipid activator protein (SAP-1), Arch. Biochem. Biophys. 233: 556 (1984).CrossRefGoogle Scholar
  24. 24.
    E. Neufeld, Recognition and processing of lysosomal enzymes in cultured fibroblasts, in “Lysosomes and Lysosomal Storage Diseases,” J.W. Callahan and J.A. Lowden, eds., Raven Press, New York (1981).Google Scholar
  25. 25.
    V. Gieselmann, R. Pohlmann, A. Hasilik, and K. Von Figura, Biosynthesis and transport of cathepsin D in cultured human fibroblasts, J. Cell Biol. 97: 1 (1983).PubMedCrossRefGoogle Scholar
  26. 26.
    S. Fujibayashi, F.-T. Kao, C. Jones, H. Morse, M. Law, and D.A. Wenger, Assignment of the gene for human sphingolipid activator protein-2 (SAP-2) to chromosome 10, Am. J. Hum. Genet. 37: 741 (1985).PubMedGoogle Scholar
  27. 27.
    K. Inui, F.-T. Kao, S. Fujibayashi, C. Jones, H.G. Morse, M.L. Law, and D.A. Wenger, The gene coding for a sphingolipid activator protein, SAP-1, is on human chromosome 10. Hum. Genet. 69: 197 (1985).PubMedCrossRefGoogle Scholar
  28. 28.
    U. Gubler, P. Seeberg, B.J. Hoffman, L.P. Gage and S. Udenfriend, Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides, Nature 295: 206 (1982).PubMedCrossRefGoogle Scholar
  29. 29.
    N. Dewji, D. Wenger, S. Fujibayashi, M. Donoviel, F. Esch, F. Hill, and J.S. O’Brien, Molecular cloning of sphingolipid activator protein 1 (SAP-1), the sulfatide sulfatase activator, Am. J. Hum. Genet. 37: A150 (1985).Google Scholar

Copyright information

© Plenum Press, New York 1986

Authors and Affiliations

  • David A. Wenger
    • 1
  • Shinsuke Fujibayashi
    • 1
  1. 1.Department of PediatricsUniversity of Colorado Health Sciences CenterDenverUSA

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