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J. Biol. Chem., Vol. 281, Issue 43, 32451-32460, October 27, 2006

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Saposin A Mobilizes Lipids from Low Cholesterol and High Bis(monoacylglycerol)phosphate-containing Membranes

PATIENT VARIANT SAPOSIN A LACKS LIPID EXTRACTION CAPACITY^*

Silvia Locatelli-Hoops, Natascha Remmel, Ralf Klingenstein, Bernadette Breiden, Maksim Rossocha, Maike Schoeniger, Christine Koenigs, Wolfram Saenger, and Konrad Sandhoff¹

From the Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany and Institut für Chemie, Abteilung Kristallographie, Freie Universität Berlin, Takustrasse 6, D-14195 Berlin, Germany

Saposin A (Sap-A) is one of five known sphingolipid activator proteins required for the lysosomal degradation of sphingolipids and for the loading of lipid antigens onto antigen-presenting molecules of the CD1 type. Sap-A assists in the degradation of galactosylceramide by galactosylceramide--galactosidase in vivo, which takes place at the surface of intraendosomal/intralysosomal vesicles. Sap-A is believed to mediate the interaction between the enzyme and its membrane-bound substrate. Its dysfunction causes a variant form of Krabbe disease. In the present study we prepared glycosylated Sap-A free of other Saps, taking advantage of the Pichia pastoris expression system. Using liposomes and surface plasmon resonance spectroscopy, we tested the binding and lipid mobilization capacity of Sap-A under different conditions. Along the endocytic pathway, the pH value decreases, and the lipid composition of intraendosomal and intralysosomal membranes changes drastically. In the inner membranes the cholesterol concentration decreases, and that of the anionic phospholipid bis(monoacylglycero)phosphate increases. Here, we show that Sap-A is able to bind to liposomes and to mobilize lipids out of them at acidic pH values below pH 4.7. Low cholesterol levels and increasing concentrations of bis(monoacylglycero)phosphate favor lipid extraction significantly. Galactosylceramide as a bilayer component is not essential for lipid mobilization by Sap-A, which requires intact disulfide bridges for activity. We also show for the first time that glycosylation of Sap-A is essential for its lipid extraction activity. Variant Sap-A proteins, which cause storage of galactosylceramide in humans (Krabbe disease, Spiegel, R., Bach, G., Sury, V., Mengistu, G., Meidan, B., Shalev, S., Shneor, Y., Mandel, H., and Zeigler, M. (2005) Mol. Genet. Metab. 84, 160–166) and in mutant mice (Matsuda, J., Vanier, M. T., Saito, Y., Tohyama, J., and Suzuki, K. (2001) Hum. Mol. Genet. 10, 1191–1199) are deficient in lipid extraction capacity.

Received for publication, August 1, 2006

^* This work was supported by Deutsche Forschungsgemeinschaft Grant Sa 257/21, SFB 645 and the Bundesministerium für Bildung und Forschung (Leukonet). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Kekulé-Institut für Organische Chemie und Biocheme, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany. Tel.: 49-228-735346; Fax: 49-228-737778; E-mail: sandhoff{at}uni-bonn.de

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