Saposin B mobilizes lipids from cholesterol‐poor and bis(monoacylglycero)phosphate‐rich membranes at acidic pH

Sphingolipid activator proteins (SAPs), GM2 activator protein (GM2AP) and saposins (Saps) A–D are small, enzymatically inactive glycoproteins of the lysosome. Despite of their sequence homology, these lipid‐binding and ‐transfer proteins show different specificities and varying modes of action. Water‐soluble SAPs facilitate the degradation of membrane‐bound glycosphingolipids with short oligosaccharide chains by exohydrolases at the membrane–water interface. There is strong evidence that degradation of endocytosed components of the cell membrane takes place at intraendosomal and intralysosomal membranes. The inner membranes of the lysosome differ from the limiting membrane of the organelle in some typical ways: the inner vesicular membranes lack a protecting glycocalix, and they are almost free of cholesterol, but rich in bis(monoacylglycero)phosphate (BMP), the anionic marker lipid of lysosomes. In this study, we prepared glycosylated Sap‐B free of other Saps by taking advantage of the Pichia pastoris expression system. We used immobilized liposomes as a model for intralysosomal vesicular membranes to probe their interaction with recombinantly expressed Sap‐B. We monitored this interaction using SPR spectroscopy and an independent method based on the release of radioactively labelled lipids from liposomal membranes. We show that, after initial binding, Sap‐B disturbs the membrane structure and mobilizes the lipids from it. Lipid mobilization is dependent on an acidic pH and the presence of anionic lipids, whereas cholesterol is able to stabilize the liposomes. We also show for the first time that glycosylation of Sap‐B is essential to achieve its full lipid‐extraction activity. Removal of the carbohydrate moiety of Sap‐B reduces its membrane‐destabilizing quality. An unglycosylated Sap‐B variant, Asn215His, which causes a fatal sphingolipid storage disease, lost the ability to extract membrane lipids at acidic pH in the presence of BMP.

[1]  M. Wendeler,et al.  Development of an assay for the intermembrane transfer of cholesterol by Niemann-Pick C2 protein , 2007, Biological chemistry.

[2]  M. Patterson,et al.  PART 16 : LYSOSOMAL DISORDERS Chapter 145 : Niemann-Pick Disease Type C : A Lipid Trafficking Disorder , 2007 .

[3]  Ralf Klingenstein,et al.  Saposin A Mobilizes Lipids from Low Cholesterol and High Bis(monoacylglycerol)phosphate-containing Membranes , 2006, Journal of Biological Chemistry.

[4]  J. Storch,et al.  Mechanism of Cholesterol Transfer from the Niemann-Pick Type C2 Protein to Model Membranes Supports a Role in Lysosomal Cholesterol Transport*♦ , 2006, Journal of Biological Chemistry.

[5]  C. Yip,et al.  Direct visualization of saposin remodelling of lipid bilayers. , 2006, Journal of molecular biology.

[6]  G. Privé,et al.  Crystal structures of saposins A and C , 2006, Protein science : a publication of the Protein Society.

[7]  S. Sonnino,et al.  Saposin B binds and transfers phospholipids Published, JLR Papers in Press, February 6, 2006. , 2006, Journal of Lipid Research.

[8]  Thomas Kolter,et al.  The enzyme‐binding region of human GM2‐activator protein , 2006, The FEBS journal.

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

[10]  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.

[11]  H. Bruhn.  A short guided tour through functional and structural features of saposin-like proteins. , 2005, The Biochemical journal.

[12]  M. Elleder,et al.  Prosaposin deficiency -- a rarely diagnosed, rapidly progressing, neonatal neurovisceral lipid storage disease. Report of a further patient. , 2005, Neuropediatrics.

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

[14]  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.

[15]  R. Proia,et al.  Lysosomal Glycosphingolipid Recognition by NKT Cells , 2004, Science.

[16]  Analysis of recombinant human saposin A expressed by Pichia pastoris. , 2004, Biochemical and biophysical research communications.

[17]  Horst Kessler,et al.  Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization. , 2004, Protein expression and purification.

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

[19]  K. Sandhoff,et al.  A non-glycosylated and functionally deficient mutant (N215H) of the sphingolipid activator protein B (SAP-B) in a novel case of metachromatic leukodystrophy (MLD) , 2000, Journal of Inherited Metabolic Disease.

[20]  R. Salvioli,et al.  Saposins and Their Interaction with Lipids , 1999, Neurochemical Research.

[21]  N. Packer,et al.  A general approach to desalting oligosaccharides released from glycoproteins , 1998, Glycoconjugate Journal.

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

[23]  D. Wenger,et al.  The mechanism for a 33-nucleotide insertion in mRNA causing sphingolipid activator protein (SAP-1)-deficient metachromatic leukodystrophy , 1991, Human Genetics.

[24]  A. Waring,et al.  Characterization of a recombinant molecule covalently indistinguishable from human cerebroside-sulfate activator protein (CSAct or Saposin B). , 2003, Cellular and molecular biology.

[25]  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.

[26]  P. Saftig,et al.  At the acidic edge: emerging functions for lysosomal membrane proteins. , 2003, Trends in cell biology.

[27]  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.

[28]  G. Privé,et al.  Crystal structure of saposin B reveals a dimeric shell for lipid binding , 2002, Proceedings of the National Academy of Sciences of the United States of America.

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

[30]  K. Sandhoff,et al.  Sphingolipid activator proteins: proteins with complex functions in lipid degradation and skin biogenesis. , 2001, Glycobiology.

[31]  M. Elleder,et al.  A novel mutation in the coding region of the prosaposin gene leads to a complete deficiency of prosaposin and saposins, and is associated with a complex sphingolipidosis dominated by lactosylceramide accumulation. , 2001, Human molecular genetics.

[32]  J. Whitelegge,et al.  Comparative lipid binding study on the cerebroside sulfate activator (saposin B) , 2001, Journal of neuroscience research.

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

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

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

[36]  A. Blume,et al.  Interaction of the GM2-activator protein with phospholipid-ganglioside bilayer membranes and with monolayers at the air-water interface. , 2001, European journal of biochemistry.

[37]  S. Regis,et al.  An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity , 1999, European Journal of Human Genetics.

[38]  D. Williams,et al.  Surface plasmon resonance analysis at a supported lipid monolayer. , 1998, Biochimica et biophysica acta.

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

[40]  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.

[41]  A. Brown,et al.  Transacylase and phospholipases in the synthesis of bis(monoacylglycero)phosphate. , 1996, Biochemistry.

[42]  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.

[43]  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.

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

[45]  R. Munford,et al.  Saposin-like proteins (SAPLIP) carry out diverse functions on a common backbone structure. , 1995, Journal of lipid research.

[46]  K. von Figura,et al.  Biogenesis of lysosomal membranes , 1994, FEBS letters.

[47]  K. Sandhoff,et al.  Sphingolipid activator protein D (sap-D) stimulates the lysosomal degradation of ceramide in vivo. , 1994, Biochemical and biophysical research communications.

[48]  G. Schwarzmann,et al.  Hydrolysis of lactosylceramide by human galactosylceramidase and GM1-beta-galactosidase in a detergent-free system and its stimulation by sphingolipid activator proteins, sap-B and sap-C. Activator proteins stimulate lactosylceramide hydrolysis. , 1994, European journal of biochemistry.

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

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

[51]  B. Martin,et al.  The effect of carbohydrate removal on stability and activity of saposin B. , 1993, Archives of biochemistry and biophysics.

[52]  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.

[53]  K. Sandhoff,et al.  Glycosphingolipid specificity of the human sulfatide activator protein. , 1991, European journal of biochemistry.

[54]  B. Persson,et al.  Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins , 1991 .

[55]  K. Sandhoff,et al.  Sulfatide activator protein. Alternative splicing that generates three mRNAs and a newly found mutation responsible for a clinical disease. , 1991, The Journal of biological chemistry.

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

[57]  K. Kretz,et al.  Characterization of a mutation in a family with saposin B deficiency: a glycosylation site defect. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

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

[59]  H. Hauser,et al.  Vectors for efficient expression in mammalian fibroblastoid, myeloid and lymphoid cells via transfection or infection. , 1988, Gene.

[60]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[61]  P. K. Smith,et al.  Measurement of protein using bicinchoninic acid. , 1985, Analytical biochemistry.

[62]  K. Sandhoff,et al.  Activator protein for the degradation of globotriaosylceramide by human alpha-galactosidase. , 1983, The Journal of biological chemistry.

[63]  K. Sandhoff,et al.  Complexing of glycolipids and their transfer between membranes by the activator protein for degradation of lysosomal ganglioside GM2. , 1982, European journal of biochemistry.

[64]  H. Heidrich,et al.  The phospholipids of liver lysosomes from untreated rats. , 1980, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[65]  Y. Matsuzawa,et al.  Degradation of bis(monoacylglycero)phosphate by an acid phosphodiesterase in rat liver lysosomes. , 1979, The Journal of biological chemistry.

[66]  G. Fischer,et al.  Enzymic hydrolysis of sulphosphingolipids and sulphoglycerolipids by sulphatase A in the presence and absence of activator protein. , 1978, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[67]  G. Fischer,et al.  The activator of cerebroside-sulphatase. A model of the activation. , 1978, Biochimica et biophysica acta.

[68]  W. Stremmel,et al.  [Bis(monoacylglycero)phosphate--a marker lipid of secondary lysosomes? (AUTHOR'S TRANSL)]. , 1976, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

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

[70]  H. Jatzkewitz,et al.  Comparison of the cerebroside sulphatase and the arylsulphatase activity of human sulphatase A in the absence of activators. , 1975, Biochimica et biophysica acta.

[71]  G. Fischer,et al.  The activator of cerebroside sulphatase. Purification from human liver and identification as a protein. , 1975, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.