Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 284, Issue 14, Pages 9372-9381Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M809457200
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Funding
- European Leukodystrophy Association
- Bundesministerium fur Bildung und Forschung (Leukonet)
- Deutsche Forschungsgemeinschaft [Sa 257/21, SFB 645, 202272]
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Arylsulfatase A (ASA) catalyzes the intralysosomal desulfation of 3-O-sulfogalactosylceramide (sulfatide) to galactosylceramide. The reaction requires saposin B (Sap B), a non-enzymatic protein-aceous cofactor which presents sulfatide to the catalytic site of ASA. The lack of either ASA or Sap B results in a block of sulfatide degradation, progressive intralysosomal accumulation of sulfatide, and the fatal lysosomal storage disease metachromatic leukodystrophy. We studied the coupled Sap B-ASA reaction in vitro using detergent-free micellar and liposomal assay systems and in vivo using cell culture models of metachromatic leukodystrophy. Under in vitro conditions, the reaction had a narrowpHoptimum around pH 4.3 and was inhibited by mono- and divalent cations, phosphate and sulfite. Bis(monoacylglycero) phosphate and phosphatidic acid were activators of the reaction, underscoring a significant role of acidic phosphoglycerolipids in sphingolipid degradation. Desulfation was negligible when Sap B was substituted by Sap A, C, or D. Up to a molar ratio between Sap B and sulfatide of 1: 5, an elevation of Sap B concentrations caused a sharp increase of sulfatide hydrolysis, indicating the requirement of unexpected high Sap B levels for maximum turnover. Feeding of ASA-deficient, sulfatide-storing primary mouse kidney cells with ASA caused partial clearance of sulfatide. Co-feeding of Sap B or its precursor prosaposin resulted in the lysosomal uptake of the cofactor but did not promote ASA-catalyzed sulfatide hydrolysis. This suggests that Sap B is not a limiting factor of the coupled Sap B-ASA reaction in mouse kidney cells even if sulfatide has accumulated to unphysiologically high levels.
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