title: Saposin B-dependent reconstitution of arylsulfatase A activity in vitro and in cell culture models of metachromatic leukodystrophy.
abstract: Arylsulfatase A (ASA) catalyzes the intralysosomal desulfation of 3-O-sulfogalactosylceramide (sulfatide) to galactosylceramide. The reaction requires saposin B (Sap B), a non-enzymatic proteinaceous 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 narrow pH optimum 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.
authors.affiliation: Institut für Physiologische Chemie and LIMES, Membrane Biology and Lipid Biochemistry Unit, c/o Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-University, 53115 Bonn, Germany. matzner@institut.physiochem.uni-bonn.de
authors.forename: Ulrich
authors.initials: U
authors.lastname: Matzner
authors.forename: Bernadette
authors.initials: B
authors.lastname: Breiden
authors.forename: Günter
authors.initials: G
authors.lastname: Schwarzmann
authors.forename: Afshin
authors.initials: A
authors.lastname: Yaghootfam
authors.forename: Arvan L
authors.initials: AL
authors.lastname: Fluharty
authors.forename: Andrej
authors.initials: A
authors.lastname: Hasilik
authors.forename: Konrad
authors.initials: K
authors.lastname: Sandhoff
authors.forename: Volkmar
authors.initials: V
authors.lastname: Gieselmann
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002553:Cerebroside-Sulfatase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D007966:Leukodystrophy, Metachromatic / Q000201:enzymology* / Q000235:genetics; D050356:Lipid Metabolism; D008081:Liposomes; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D049231:Saposins / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
chemical_list: D004220:Disulfides; D008081:Liposomes; D049231:Saposins; D002553:Cerebroside-Sulfatase