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. 2000 Sep;9(9):1618–1630. doi: 10.1110/ps.9.9.1618

Methionine oxidation within the cerebroside-sulfate activator protein (CSAct or Saposin B).

J P Whitelegge 1, B Penn 1, T To 1, J Johnson 1, A Waring 1, M Sherman 1, R L Stevens 1, C B Fluharty 1, K F Faull 1, A L Fluharty 1
PMCID: PMC2144706  PMID: 11045609

Abstract

The cerebroside-sulfate activator protein (CSAct or Saposin B) is a small water-soluble glycoprotein that plays an essential role in the metabolism of certain glycosphingolipids, especially sulfatide. Deficiency of CSAct in humans leads to sulfatide accumulation and neurodegenerative disease. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A. CSAct has seven methionine residues and a mass of 8,845 Da when deglycosylated. Mildly oxidized, deglycosylated CSAct (+16 Da), separated from nonoxidized CSAct by reversed-phase high-performance liquid chromatography (RP-HPLC), showed significant modulation of the in vitro activity. Because oxidation partially protected against CNBr cleavage and could largely be reversed by treatment with dithiothreitol, it was concluded that the major modification was conversion of a single methionine to its sulfoxide. High-resolution RP-HPLC separated mildly oxidized CSAct into seven or more different components with shorter retention times than nonoxidized CSAct. Mass spectrometry showed these components to have identical mass (+16 Da). The shorter retention times are consistent with increased polarity accompanying oxidation of surface-exposed methionyl side chains, in general accordance with the existing molecular model. A mass-spectrometric CNBr mapping protocol allowed identification of five of the seven possible methionine-sulfoxide CSAct oxoforms. The most dramatic suppression of activity occurred upon oxidation of Met61 (26% of control) with other residues in the Q60MMMHMQ66 motif falling in the 30-50% activity range. Under conditions of oxidative stress, accumulation of minimally oxidized CSAct protein in vivo could perturb metabolism of sulfatide and other glycosphingolipids. This, in turn, could contribute to the onset and progression of neurodegenerative disease, especially in situations where the catabolism of these materials is marginal.

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Selected References

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  1. Anantharamaiah G. M., Hughes T. A., Iqbal M., Gawish A., Neame P. J., Medley M. F., Segrest J. P. Effect of oxidation on the properties of apolipoproteins A-I and A-II. J Lipid Res. 1988 Mar;29(3):309–318. [PubMed] [Google Scholar]
  2. Azuma N., Seo H. C., Lie O., Fu Q., Gould R. M., Hiraiwa M., Burt D. W., Paton I. R., Morrice D. R., O'Brien J. S. Cloning, expression and map assignment of chicken prosaposin. Biochem J. 1998 Feb 15;330(Pt 1):321–327. doi: 10.1042/bj3300321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berti P. J., Ekiel I., Lindahl P., Abrahamson M., Storer A. C. Affinity purification and elimination of methionine oxidation in recombinant human cystatin C. Protein Expr Purif. 1997 Oct;11(1):111–118. doi: 10.1006/prep.1997.0763. [DOI] [PubMed] [Google Scholar]
  4. Chao C. C., Ma Y. S., Stadtman E. R. Modification of protein surface hydrophobicity and methionine oxidation by oxidative systems. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2969–2974. doi: 10.1073/pnas.94.7.2969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collard M. W., Sylvester S. R., Tsuruta J. K., Griswold M. D. Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator. Biochemistry. 1988 Jun 14;27(12):4557–4564. doi: 10.1021/bi00412a050. [DOI] [PubMed] [Google Scholar]
  6. Conzelmann E., Sandhoff K. Biochemical basis of late-onset neurolipidoses. Dev Neurosci. 1991;13(4-5):197–204. doi: 10.1159/000112160. [DOI] [PubMed] [Google Scholar]
  7. Dauber-Osguthorpe P., Roberts V. A., Osguthorpe D. J., Wolff J., Genest M., Hagler A. T. Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system. Proteins. 1988;4(1):31–47. doi: 10.1002/prot.340040106. [DOI] [PubMed] [Google Scholar]
  8. Dewji N., Wenger D., Fujibayashi S., Donoviel M., Esch F., Hill F., O'Brien J. S. Molecular cloning of the sphingolipid activator protein-1 (SAP-1), the sulfatide sulfatase activator. Biochem Biophys Res Commun. 1986 Jan 29;134(2):989–994. doi: 10.1016/s0006-291x(86)80518-6. [DOI] [PubMed] [Google Scholar]
  9. Faull K. F., Higginson J., Waring A. J., Johnson J., To T., Whitelegge J. P., Stevens R. L., Fluharty C. B., Fluharty A. L. Disulfide connectivity in cerebroside sulfate activator is not necessary for biological activity or alpha-helical content but is necessary for trypsin resistance and strong ligand binding. Arch Biochem Biophys. 2000 Apr 15;376(2):266–274. doi: 10.1006/abbi.2000.1714. [DOI] [PubMed] [Google Scholar]
  10. Faull K. F., Higginson J., Waring A. J., To T., Whitelegge J. P., Stevens R. L., Fluharty C. B., Fluharty A. L. Hydrogen-deuterium exchange signature of porcine cerebroside sulfate activator protein. J Mass Spectrom. 2000 Mar;35(3):392–401. doi: 10.1002/(SICI)1096-9888(200003)35:3<392::AID-JMS948>3.0.CO;2-T. [DOI] [PubMed] [Google Scholar]
  11. Faull K. F., Whitelegge J. P., Higginson J., To T., Johnson J., Krutchinsky A. N., Standing K. G., Waring A. J., Stevens R. L., Fluharty C. B. Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties. J Mass Spectrom. 1999 Oct;34(10):1040–1054. doi: 10.1002/(SICI)1096-9888(199910)34:10<1040::AID-JMS863>3.0.CO;2-X. [DOI] [PubMed] [Google Scholar]
  12. Fischer G., Jatzkewitz H. The activator of cerebroside sulphatase. Binding studies with enzyme and substrate demonstrating the detergent function of the activator protein. Biochim Biophys Acta. 1977 Apr 12;481(2):561–572. doi: 10.1016/0005-2744(77)90288-1. [DOI] [PubMed] [Google Scholar]
  13. Fischer G., Reiter S., Jatzkewitz H. Enzymic hydrolysis of sulphosphingolipids and sulphoglycerolipids by sulphatase A in the presence and absence of activator protein. Hoppe Seylers Z Physiol Chem. 1978 Jul;359(7):863–866. [PubMed] [Google Scholar]
  14. Fluharty A. L., Davis M. L., Kihara H., Kritchevsky G. Simplified procedure for preparation of 35S-labeled brain sulfatide. Lipids. 1974 Nov;9(11):865–869. doi: 10.1007/BF02532611. [DOI] [PubMed] [Google Scholar]
  15. Fluharty A. L., Katona Z., Meek W. E., Frei K., Fowler A. V. The cerebroside sulfate activator from pig kidney: purification and molecular structure. Biochem Med Metab Biol. 1992 Feb;47(1):66–85. doi: 10.1016/0885-4505(92)90009-n. [DOI] [PubMed] [Google Scholar]
  16. Fluharty A. L., Lombardo C., Louis A., Stevens R. L., Whitelegge J., Waring A. J., To T., Fluharty C. B., Faull K. F. Preparation of the cerebroside sulfate activator (CSAct or saposin B) from human urine. Mol Genet Metab. 1999 Nov;68(3):391–403. doi: 10.1006/mgme.1999.2900. [DOI] [PubMed] [Google Scholar]
  17. Fluharty A. L., Meek W. E., Katona Z., Tsay K. K. The cerebroside sulfate activator from pig kidney: derivitization, cerebroside sulfate binding, and metabolic correction. Biochem Med Metab Biol. 1992 Feb;47(1):86–96. doi: 10.1016/0885-4505(92)90010-v. [DOI] [PubMed] [Google Scholar]
  18. Gao J., Yin D., Yao Y., Williams T. D., Squier T. C. Progressive decline in the ability of calmodulin isolated from aged brain to activate the plasma membrane Ca-ATPase. Biochemistry. 1998 Jun 30;37(26):9536–9548. doi: 10.1021/bi9803877. [DOI] [PubMed] [Google Scholar]
  19. Garner B., Waldeck A. R., Witting P. K., Rye K. A., Stocker R. Oxidation of high density lipoproteins. II. Evidence for direct reduction of lipid hydroperoxides by methionine residues of apolipoproteins AI and AII. J Biol Chem. 1998 Mar 13;273(11):6088–6095. doi: 10.1074/jbc.273.11.6088. [DOI] [PubMed] [Google Scholar]
  20. Holtschmidt H., Sandhoff K., Fürst W., Kwon H. Y., Schnabel D., Suzuki K. The organization of the gene for the human cerebroside sulfate activator protein. FEBS Lett. 1991 Mar 25;280(2):267–270. doi: 10.1016/0014-5793(91)80308-p. [DOI] [PubMed] [Google Scholar]
  21. Horstmann H. J., Rohen J. W., Sames K. Age-related changes in the composition of proteins in the trabecular meshwork of the human eye. Mech Ageing Dev. 1983 Feb;21(2):121–136. doi: 10.1016/0047-6374(83)90069-6. [DOI] [PubMed] [Google Scholar]
  22. Houghten R. A., Li C. H. Reduction of sulfoxides in peptides and proteins. Methods Enzymol. 1983;91:549–559. doi: 10.1016/s0076-6879(83)91050-9. [DOI] [PubMed] [Google Scholar]
  23. Joppich-Kuhn R., Corkill J. A., Giese R. W. Oxidation of methionine to methionine sulfoxide as a side reaction of cyanogen bromide cleavage. Anal Biochem. 1982 Jan 1;119(1):73–77. doi: 10.1016/0003-2697(82)90666-2. [DOI] [PubMed] [Google Scholar]
  24. KOSHLAND D. E., Jr, STRUMEYER D. H., RAY W. J., Jr Amino acids involved in the action of chymotrypsin. Brookhaven Symp Biol. 1962 Dec;15:101–133. [PubMed] [Google Scholar]
  25. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  26. Liaw S. H., Villafranca J. J., Eisenberg D. A model for oxidative modification of glutamine synthetase, based on crystal structures of mutant H269N and the oxidized enzyme. Biochemistry. 1993 Aug 10;32(31):7999–8003. doi: 10.1021/bi00082a022. [DOI] [PubMed] [Google Scholar]
  27. Liepinsh E., Andersson M., Ruysschaert J. M., Otting G. Saposin fold revealed by the NMR structure of NK-lysin. Nat Struct Biol. 1997 Oct;4(10):793–795. doi: 10.1038/nsb1097-793. [DOI] [PubMed] [Google Scholar]
  28. Maier K. L., Lenz A. G., Beck-Speier I., Costabel U. Analysis of methionine sulfoxide in proteins. Methods Enzymol. 1995;251:455–461. doi: 10.1016/0076-6879(95)51149-0. [DOI] [PubMed] [Google Scholar]
  29. Maple J. R., Dinur U., Hagler A. T. Derivation of force fields for molecular mechanics and dynamics from ab initio energy surfaces. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5350–5354. doi: 10.1073/pnas.85.15.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Morimoto S., Yamamoto Y., O'Brien J. S., Kishimoto Y. Distribution of saposin proteins (sphingolipid activator proteins) in lysosomal storage and other diseases. Proc Natl Acad Sci U S A. 1990 May;87(9):3493–3497. doi: 10.1073/pnas.87.9.3493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Munford R. S., Sheppard P. O., O'Hara P. J. Saposin-like proteins (SAPLIP) carry out diverse functions on a common backbone structure. J Lipid Res. 1995 Aug;36(8):1653–1663. [PubMed] [Google Scholar]
  32. Nakano T., Sandhoff K., Stümper J., Christomanou H., Suzuki K. Structure of full-length cDNA coding for sulfatide activator, a Co-beta-glucosidase and two other homologous proteins: two alternate forms of the sulfatide activator. J Biochem. 1989 Feb;105(2):152–154. doi: 10.1093/oxfordjournals.jbchem.a122629. [DOI] [PubMed] [Google Scholar]
  33. Ota I. M., Clarke S. Enzymatic methylation of L-isoaspartyl residues derived from aspartyl residues in affinity-purified calmodulin. The role of conformational flexibility in spontaneous isoaspartyl formation. J Biol Chem. 1989 Jan 5;264(1):54–60. [PubMed] [Google Scholar]
  34. Spector A., Scotto R., Weissbach H., Brot N. Lens methionine sulfoxide reductase. Biochem Biophys Res Commun. 1982 Sep 16;108(1):429–434. doi: 10.1016/0006-291x(82)91884-8. [DOI] [PubMed] [Google Scholar]
  35. Stadtman E. R. Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu Rev Biochem. 1993;62:797–821. doi: 10.1146/annurev.bi.62.070193.004053. [DOI] [PubMed] [Google Scholar]
  36. Stadtman E. R. Protein oxidation and aging. Science. 1992 Aug 28;257(5074):1220–1224. doi: 10.1126/science.1355616. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Stevens R. L., Fluharty A. L., Kihara H., Kaback M. M., Shapiro L. J., Marsh B., Sandhoff K., Fischer G. Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy. Am J Hum Genet. 1981 Nov;33(6):900–906. [PMC free article] [PubMed] [Google Scholar]
  39. Tsuda M., Sakiyama T., Endo H., Kitagawa T. The primary structure of mouse saposin. Biochem Biophys Res Commun. 1992 May 15;184(3):1266–1272. doi: 10.1016/s0006-291x(05)80019-1. [DOI] [PubMed] [Google Scholar]
  40. Vaccaro A. M., Salvioli R., Barca A., Tatti M., Ciaffoni F., Maras B., Siciliano R., Zappacosta F., Amoresano A., Pucci P. Structural analysis of saposin C and B. Complete localization of disulfide bridges. J Biol Chem. 1995 Apr 28;270(17):9953–9960. doi: 10.1074/jbc.270.17.9953. [DOI] [PubMed] [Google Scholar]
  41. Van Patten S. M., Hanson E., Bernasconi R., Zhang K., Manavalan P., Cole E. S., McPherson J. M., Edmunds T. Oxidation of methionine residues in antithrombin. Effects on biological activity and heparin binding. J Biol Chem. 1999 Apr 9;274(15):10268–10276. doi: 10.1074/jbc.274.15.10268. [DOI] [PubMed] [Google Scholar]
  42. Vogel A., Fürst W., Abo-Hashish M. A., Lee-Vaupel M., Conzelmann E., Sandhoff K. Identity of the activator proteins for the enzymatic hydrolysis of sulfatide, ganglioside GM1, and globotriaosylceramide. Arch Biochem Biophys. 1987 Dec;259(2):627–638. doi: 10.1016/0003-9861(87)90529-7. [DOI] [PubMed] [Google Scholar]
  43. Vogt W. Oxidation of methionyl residues in proteins: tools, targets, and reversal. Free Radic Biol Med. 1995 Jan;18(1):93–105. doi: 10.1016/0891-5849(94)00158-g. [DOI] [PubMed] [Google Scholar]
  44. Waring A. J., Chen Y., Faull K. F., Stevens R., Sherman M. A., Fluharty A. L. Porcine cerebroside sulfate activator (saposin B) secondary structure: CD, FTIR, and NMR studies. Mol Genet Metab. 1998 Jan;63(1):14–25. doi: 10.1006/mgme.1997.2646. [DOI] [PubMed] [Google Scholar]
  45. Whitelegge J. P., Gundersen C. B., Faull K. F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins. Protein Sci. 1998 Jun;7(6):1423–1430. doi: 10.1002/pro.5560070619. [DOI] [PMC free article] [PubMed] [Google Scholar]

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