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

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-β-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.

[1]  N. Radin,et al.  Cerebroside galactosidase of brain. , 1966, Journal of lipid research.

[2]  M. Ho,et al.  Gaucher's disease: deficiency of 'acid' -glucosidase and reconstitution of enzyme activity in vitro. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Wherrett,et al.  Enrichment of bis-(monoacylglyceryl) phosphate in lysosomes from rat liver. , 1972, The Journal of biological chemistry.

[4]  W. Stremmel,et al.  Bis(monoacylglycerin)phosphorsäure — ein Marker-Lipid sekundärer Lysosomen? , 1976 .

[5]  N. Radin,et al.  Mechanism of activation of glucocerebrosidase by co-beta-glucosidase (glucosidase activator protein). , 1981, Biochimica et biophysica acta.

[6]  S. Roth,et al.  A protein activator of galactosylceramide β-galactosidase , 1982 .

[7]  R. Linke,et al.  Immunochemical characterization of two activator proteins stimulating enzymic sphingomyelin degradation in vitro. Absence of one of them in a human Gaucher disease variant. , 1986, Biological chemistry Hoppe-Seyler.

[8]  K. Sandhoff,et al.  Specificity of human glucosylceramide β‐glucosidase towards synthetic glucosylsphingolipids inserted into liposomes , 1986 .

[9]  I Mellman,et al.  Acidification of the endocytic and exocytic pathways. , 1986, Annual review of biochemistry.

[10]  B. Martin,et al.  Saposin A: second cerebrosidase activator protein. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[11]  S. Weiler,et al.  Interaction of saposins, acidic lipids, and glucosylceramidase. , 1990, The Journal of biological chemistry.

[12]  G. Grabowski,et al.  Human acid beta-glucosidase. Use of inhibitory and activating monoclonal antibodies to investigate the enzyme's catalytic mechanism and saposin A and C binding sites. , 1991, The Journal of biological chemistry.

[13]  R. Macdonald,et al.  Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. , 1991, Biochimica et biophysica acta.

[14]  K. Sandhoff,et al.  Mutation in the sphingolipid activator protein 2 in a patient with a variant of Gaucher disease , 1991, FEBS letters.

[15]  Y. Kishimoto,et al.  Saposins: structure, function, distribution, and molecular genetics. , 1992, Journal of lipid research.

[16]  A. Poulos,et al.  Simultaneous deficiency of sphingolipid activator proteins 1 and 2 is caused by a mutation in the initiation codon of their common gene. , 1992, The Journal of biological chemistry.

[17]  B. Maras,et al.  Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes , 1993, FEBS letters.

[18]  R. Salvioli,et al.  Studies on glucosylceramidase binding to phosphatidylserine liposomes: the role of bilayer curvature. , 1993, Biochimica et biophysica acta.

[19]  C. Ponting.  Acid sphingomyelinase possesses a domain homologous to its activator proteins: Saposins B and D , 1994, Protein science : a publication of the Protein Society.

[20]  X. Qi,et al.  Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid beta-glucosidase. , 1994, The Journal of biological chemistry.

[21]  R. Salvioli,et al.  pH-dependent Conformational Properties of Saposins and Their Interactions with Phospholipid Membranes (*) , 1995, The Journal of Biological Chemistry.

[22]  E. Prence.  Effects of calcium on phosphatidylserine- and saposin C-stimulated glucosylceramide beta-glucosidase activity. , 1995, The Biochemical journal.

[23]  K. Sandhoff,et al.  Topology of glycosphingolipid degradation. , 1996, Trends in cell biology.

[24]  K. Sandhoff,et al.  Expression of the Three Alternative Forms of the Sphingolipid Activator Protein Precursor in Baby Hamster Kidney Cells and Functional Assays in a Cell Culture System (*) , 1996, The Journal of Biological Chemistry.

[25]  N. Maeda,et al.  Targeted disruption of the mouse sphingolipid activator protein gene: a complex phenotype, including severe leukodystrophy and wide-spread storage of multiple sphingolipids. , 1996, Human molecular genetics.

[26]  W. Möbius,et al.  Accumulation of sphingolipids in SAP-precursor (prosaposin)-deficient fibroblasts occurs as intralysosomal membrane structures and can be completely reversed by treatment with human SAP-precursor. , 1997, European journal of cell biology.

[27]  R. Salvioli,et al.  Effect of Saposins A and C on the Enzymatic Hydrolysis of Liposomal Glucosylceramide* , 1997, The Journal of Biological Chemistry.

[28]  T. Linke,et al.  Lysosomal Degradation on Vesicular Membrane Surfaces , 1998, The Journal of Biological Chemistry.

[29]  R. Parton,et al.  A lipid associated with the antiphospholipid syndrome regulates endosome structure and function , 1998, Nature.

[30]  T. Linke,et al.  Degradation of Membrane-bound Ganglioside GM1 , 2000, The Journal of Biological Chemistry.

[31]  R. Salvioli,et al.  Further studies on the reconstitution of glucosylceramidase activity by Sap C and anionic phospholipids , 2000, FEBS letters.

[32]  W. Cho,et al.  Mechanism of annexin I-mediated membrane aggregation. , 2000, Biochemistry.

[33]  Dudley H. Williams,et al.  A vesicle capture sensor chip for kinetic analysis of interactions with membrane-bound receptors. , 2000, Analytical biochemistry.

[34]  W. Cho,et al.  Roles of calcium ions in the membrane binding of C2 domains. , 2001, The Biochemical journal.

[35]  J. Matsuda,et al.  A mutation in the saposin A domain of the sphingolipid activator protein (prosaposin) gene results in a late-onset, chronic form of globoid cell leukodystrophy in the mouse. , 2001, Human molecular genetics.

[36]  K. Sandhoff,et al.  Degradation of membrane-bound ganglioside GM2 by beta -hexosaminidase A. Stimulation by GM2 activator protein and lysosomal lipids. , 2001, The Journal of biological chemistry.

[37]  X. Qi,et al.  Differential Membrane Interactions of Saposins A and C , 2001, The Journal of Biological Chemistry.

[38]  K. Umebayashi.  The roles of ubiquitin and lipids in protein sorting along the endocytic pathway. , 2003, Cell structure and function.

[39]  K. Harzer,et al.  Concurrent increase of cholesterol, sphingomyelin and glucosylceramide in the spleen from non‐neurologic Niemann–Pick type C patients but also patients possibly affected with other lipid trafficking disorders , 2003, FEBS letters.

[40]  E. V. van Donselaar,et al.  Recycling Compartments and the Internal Vesicles of Multivesicular Bodies Harbor Most of the Cholesterol Found in the Endocytic Pathway , 2003, Traffic.

[41]  M. Patterson.  A Riddle Wrapped in a Mystery: Understanding Niemann-Pick Disease, Type C , 2003, The neurologist.

[42]  W. Roggendorf,et al.  Prosaposin deficiency: further characterization of the sphingolipid activator protein-deficient sibs , 1993, Human Genetics.

[43]  J. Matsuda,et al.  Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse. , 2004, Human molecular genetics.

[44]  S. Kaufmann,et al.  Saposin C is required for lipid presentation by human CD1b , 2004, Nature Immunology.

[45]  M. Vanier.  Lipid Changes in Niemann-Pick Disease Type C Brain: Personal Experience and Review of the Literature , 1999, Neurochemical Research.

[46]  P. Savage,et al.  Editing of CD1d-Bound Lipid Antigens by Endosomal Lipid Transfer Proteins , 2004, Science.

[47]  A. C. Looman,et al.  Efficient expression and secretion of Aspergillus niger RH5344 polygalacturonase in Saccharomyces cerevisiae , 1995, Applied Microbiology and Biotechnology.

[48]  Direct AFM observation of saposin C-induced membrane domains in lipid bilayers: from simple to complex lipid mixtures. , 2004, Chemistry and physics of lipids.

[49]  Michael Krauss,et al.  Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2. , 2005, Molecular cell.

[50]  P. Seglen,et al.  Proteases and proteolysis in the lysosome , 1992, Experientia.

[51]  H. Mandel,et al.  A mutation in the saposin A coding region of the prosaposin gene in an infant presenting as Krabbe disease: first report of saposin A deficiency in humans. , 2005, Molecular genetics and metabolism.

[52]  A. Poulos,et al.  Sphingolipid activator protein deficiency in a 16-week-old atypical Gaucher disease patient and his fetal sibling: Biochemical signs of combined sphingolipidoses , 1989, European Journal of Pediatrics.

[53]  A. Chabás,et al.  Activator protein deficient Gaucher's disease , 1989, Klinische Wochenschrift.

[54]  K. Sandhoff,et al.  Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids. , 2005, Annual review of cell and developmental biology.

[55]  Xiaoyang Qi,et al.  Saposin C-LBPA interaction in late-endosomes/lysosomes. , 2005, Experimental cell research.

[56]  M. Leippe,et al.  Lipid-binding Proteins in Membrane Digestion, Antigen Presentation, and Antimicrobial Defense* , 2005, Journal of Biological Chemistry.