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

Published online by Cambridge University Press:  05 October 2000

JULIAN P. WHITELEGGE ,
BRANDON PENN ,
TRANG TO ,
JEFF JOHNSON ,
ALAN WARING ,
MARK SHERMAN ,
RICHARD L. STEVENS ,
CLAIRE B. FLUHARTY ,
KYM F. FAULL  and
ARVAN L. FLUHARTY
JULIAN P. WHITELEGGE
Affiliation:
Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California 90095 Department of Chemistry & Biochemistry, University of California, Los Angeles, California 90095 Department of Psychiatry & Biobehavioural Sciences, The Mental Retardation Research Center and the Neuropsychiatric Institute, University of California, Los Angeles, California 90095
BRANDON PENN
Affiliation:
Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California 90095 Department of Chemistry & Biochemistry, University of California, Los Angeles, California 90095
TRANG TO
Affiliation:
Department of Psychiatry & Biobehavioural Sciences, The Mental Retardation Research Center and the Neuropsychiatric Institute, University of California, Los Angeles, California 90095
JEFF JOHNSON
Affiliation:
Department of Psychiatry & Biobehavioural Sciences, The Mental Retardation Research Center and the Neuropsychiatric Institute, University of California, Los Angeles, California 90095
ALAN WARING
Affiliation:
Department of Pediatrics, Drew University–King Medical Center/UCLA Medical School, Los Angeles, California 90095
MARK SHERMAN
Affiliation:
Beckman Research Institute for the City of Hope, Duarte, California 91010
RICHARD L. STEVENS
Affiliation:
Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California 90095 Department of Psychiatry & Biobehavioural Sciences, The Mental Retardation Research Center and the Neuropsychiatric Institute, University of California, Los Angeles, California 90095
CLAIRE B. FLUHARTY
Affiliation:
Department of Psychiatry & Biobehavioural Sciences, The Mental Retardation Research Center and the Neuropsychiatric Institute, University of California, Los Angeles, California 90095
KYM F. FAULL
Affiliation:
Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California 90095 Department of Psychiatry & Biobehavioural Sciences, The Mental Retardation Research Center and the Neuropsychiatric Institute, University of California, Los Angeles, California 90095
ARVAN L. FLUHARTY
Affiliation:
Department of Psychiatry & Biobehavioural Sciences, The Mental Retardation Research Center and the Neuropsychiatric Institute, University of California, Los Angeles, California 90095
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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.

Keywords

neurodegenerationoxidative stressprotein structuremetachromatic leukodystrophy
Type
Research Article
Information
Protein Science , Volume 9 , Issue 9 , September 2000 , pp. 1618 - 1630
Copyright
2000 The Protein Society

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