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Methionine oxidation within the cerebroside‐sulfate activator protein (CSAct or Saposin B)
Author(s) -
Whitelegge Julian P.,
Penn Brandon,
To Trang,
Johnson Jeff,
Waring Alan,
Sherman Mark,
Stevens Richard L.,
Fluharty Claire B.,
Faull Kym F.,
Fluharty Arvan L.
Publication year - 2000
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.9.9.1618
Subject(s) - chemistry , methionine sulfoxide , cerebroside , methionine , biochemistry , sulfoxide , dithiothreitol , chromatography , in vitro , enzyme , amino acid , organic chemistry
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 dithio‐threitol, it was concluded that the major modification was conversion of a single methionine to its sulfoxide. Highresolution 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 Q 60 MMMHMQ 66 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.