title: Saposin A mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: patient variant Saposin A lacks lipid extraction capacity.
abstract: 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-beta-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.
authors.affiliation: Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany.
authors.forename: Silvia
authors.initials: S
authors.lastname: Locatelli-Hoops
authors.forename: Natascha
authors.initials: N
authors.lastname: Remmel
authors.forename: Ralf
authors.initials: R
authors.lastname: Klingenstein
authors.forename: Bernadette
authors.initials: B
authors.lastname: Breiden
authors.forename: Maksim
authors.initials: M
authors.lastname: Rossocha
authors.forename: Maike
authors.initials: M
authors.lastname: Schoeniger
authors.forename: Christine
authors.initials: C
authors.lastname: Koenigs
authors.forename: Wolfram
authors.initials: W
authors.lastname: Saenger
authors.forename: Konrad
authors.initials: K
authors.lastname: Sandhoff
mesh_terms: D002784:Cholesterol / Q000737:chemistry*; D014644:Genetic Variation; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D050356:Lipid Metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D050178:Monoglycerides / Q000737:chemistry*; D010843:Pichia / Q000235:genetics; D049231:Saposins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D050178:Monoglycerides; D049231:Saposins; D002784:Cholesterol