Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 9 de 9
Filter
Add more filters










Database
Language
Publication year range
1.
Structure ; 16(5): 809-17, 2008 May.
Article in English | MEDLINE | ID: mdl-18462685

ABSTRACT

Human saposins are essential proteins required for degradation of sphingolipids and lipid antigen presentation. Despite the conserved structural organization of saposins, their distinct modes of interaction with biological membranes are not fully understood. We describe two crystal structures of human saposin C in an "open" configuration with unusual domain swapped homodimers. This form of SapC dimer supports the "clip-on" model for SapC-induced vesicle fusion. In addition, we present the crystal structure of SapD in two crystal forms. They reveal the monomer-monomer interface for the SapD dimer, which was confirmed in solution by analytical ultracentrifugation. The crystal structure of SapD suggests that side chains of Lys10 and Arg17 are involved in initial association with the preferred anionic biological membranes by forming salt bridges with sulfate or phosphate lipid headgroups.


Subject(s)
Saposins/chemistry , Amino Acid Sequence , Cell Membrane/metabolism , Cloning, Molecular , Conserved Sequence , Crystallography, X-Ray , Cysteine/chemistry , Dimerization , Humans , Hydrophobic and Hydrophilic Interactions , Lipid Metabolism , Models, Biological , Models, Molecular , Molecular Sequence Data , Mutagenesis , Pichia/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Saposins/genetics , Saposins/isolation & purification , Sequence Homology, Amino Acid
2.
FEBS J ; 274(13): 3405-20, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17561962

ABSTRACT

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.


Subject(s)
Cholesterol/chemistry , Lipids/chemistry , Saposins/genetics , Saposins/physiology , Cell Membrane/metabolism , Cloning, Molecular , Glycosylation , Humans , Hydrogen-Ion Concentration , Liposomes/chemistry , Lysophospholipids/chemistry , Lysosomes/metabolism , Models, Biological , Monoglycerides/chemistry , Oligosaccharides/chemistry , Pichia/metabolism , Saposins/metabolism , Surface Plasmon Resonance
3.
J Biol Chem ; 281(43): 32451-60, 2006 Oct 27.
Article in English | MEDLINE | ID: mdl-16905746

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.


Subject(s)
Cholesterol/chemistry , Genetic Variation , Lipid Metabolism , Membrane Lipids/metabolism , Monoglycerides/chemistry , Saposins/metabolism , Glycosylation , Humans , Hydrogen-Ion Concentration , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Liposomes/chemistry , Liposomes/metabolism , Membrane Lipids/chemistry , Pichia/genetics , Saposins/genetics , Saposins/isolation & purification , Surface Plasmon Resonance
4.
Article in English | MEDLINE | ID: mdl-16511279

ABSTRACT

The amphiphilic saposin proteins (A, B, C and D) act at the lipid-water interface in lysosomes, mediating the hydrolysis of membrane building blocks by water-soluble exohydrolases. Human saposin C activates glucocerebrosidase and beta-galactosylceramidase. The protein has been expressed in Pichia pastoris, purified and crystallized in three different crystal forms, diffracting to a maximum resolution of 2.5 A. Hexagonal crystals grew from 2-propanol-containing solution and contain a single molecule in the asymmetric unit according to the Matthews coefficient. Orthorhombic and tetragonal crystals were both obtained with pentaerythritol ethoxylate and are predicted to contain two molecules in the asymmetric unit. Attempts to determine the respective crystal structures by molecular replacement using either the known NMR structure of human saposin C or a related crystal structure as search models have so far failed. The failure of the molecular-replacement method is attributed to conformational changes of the protein, which are known to be required for its biological activity. Crystal structures of human saposin C therefore might be the key to mapping out the conformational trajectory of saposin-like proteins.


Subject(s)
Pichia/genetics , Saposins/chemistry , Saposins/genetics , Crystallization , Crystallography, X-Ray , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
5.
J Lipid Res ; 46(10): 2254-64, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16061947

ABSTRACT

A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cerebroside sulfate activator protein (CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins (A, C, and D) and a preference for the non-hydroxylated species of the sulfatide substrate over the corresponding hydroxylated species. The modified assay for the coupled ASA/CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.


Subject(s)
Cerebroside-Sulfatase/analysis , Saposins/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Cattle , Lithium/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
6.
Asian J Androl ; 7(2): 147-58, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15897971

ABSTRACT

AIM: To determine the effect of saposin C (a known trophic domain of prosaposin) on proliferation, migration and invasion, as well as its effect on the expression of urokinase plasmonogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMP)-2 and -9 in normal and malignant prostate cells. In addition, we tested whether saposin C can activate p42/44 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signal transduction pathways of the mitogen-activated protein kinase (MAPK) superfamily. METHODS: We employed Western blot analysis, phospho-specific antibodies, cell proliferation assay, reverse transcriptase-polymerase chain reaction, in vitro kinase assays and migration and invasion to determine the effect of saposin C on various biological behaviors of prostate stromal and cancer cells. RESULTS: Saposin C, in a cell type-specific manner, upregulates uPA/uPAR and immediate early gene c-Jun expression, stimulates cell proliferation, migration and invasion and activates p42/44 and SAPK/JNK MAPK pathways in prostate stromal and cancer cells. Normal prostate epithelial cells were not responsive to saposin C treatment in the above studies. CONCLUSION: Saposin C functions as a multipotential modulator of diverse biological activities in prostate cancer and stromal cells. These results strongly suggest that saposin C functions as a potent growth factor for prostatic cells and may contribute to prostate carcinogenesis and/or the development of hormone-refractory prostate cancer.


Subject(s)
Cell Division/drug effects , Mitogen-Activated Protein Kinases/metabolism , Neoplasm Invasiveness , Prostatic Neoplasms/metabolism , Receptors, Cell Surface/genetics , Saposins/pharmacology , Signal Transduction , Stromal Cells/metabolism , Up-Regulation , Urokinase-Type Plasminogen Activator/genetics , Enzyme Activation , Humans , Male , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Receptors, Urokinase Plasminogen Activator , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/enzymology , Stromal Cells/pathology
7.
Prostate ; 61(2): 114-23, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15305334

ABSTRACT

BACKGROUND: Prosaposin is a neurotrophic factor. Prosaposin knock-out mice have been reported to develop a number of abnormalities, including atrophy of the prostate gland and mitogen-activated protein kinase (MAPK)-inactivation in prostate epithelial cells. These abnormalities underscore a potential fundamental role in prostate development. The trophic factor activity of prosaposin has been localized at a specific amino terminal portion of the molecule that has been the source for a number of biologically active peptides called prosaptides (e.g., TX14A). The expression and function of prosaposin in prostate cancer is not known. METHODS: Using conventional protein expression analysis, immunohistochemical staining, cell proliferation assays, and in vitro invasion assays, we determined the expression of prosaposin and the effect of prosaptide TX14A on cell growth/death protection, motility, invasion, and MAPK signal transduction pathway in prostate cancer cells. RESULTS: We found a higher expression of prosaposin in androgen-independent (AI) prostate cancer cells (PC-3 and DU-145) than in androgen-dependent (AD) LNCaP or normal prostate epithelial cells. Immunohistochemical staining on benign and malignant prostate tissues revealed an intense cytoplasmic anti-prosaposin immunoreactivity in tumor cells, as well as stromal, endothelial, and inflammatory mononuclear cells. The intensity of staining was proportional to the overall Gleason's score. In addition, we demonstrated that TX14A stimulates cell proliferation/survival, migration, and invasion, and activates the Raf-MEK-ERK-RSK-Elk-1 signaling cascade of the MAPK pathway. CONCLUSIONS: These results are suggestive of a potential pleuripotent regulatory function for prosaposin in prostate cancer.


Subject(s)
Cell Movement/drug effects , Glycoproteins/metabolism , MAP Kinase Signaling System/drug effects , Nerve Growth Factors/pharmacology , Prostatic Neoplasms/physiopathology , Cell Division/drug effects , Cell Line, Tumor , Humans , Male , Neoplasm Invasiveness/physiopathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Saposins
8.
Nat Immunol ; 5(2): 169-74, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14716313

ABSTRACT

Lipids from Mycobacterium tuberculosis are presented through CD1 proteins to T lymphocytes in humans, but the accessory molecules required for antigen loading and presentation remain unidentified. Here we show that fibroblasts deficient in sphingolipid activator proteins (SAPs) transfected with CD1b failed to activate lipid-specific T cells. However, the T cell response was restored when fibroblasts were reconstituted with SAP-C but not other SAPs. Lipid antigen and SAP-C colocalized in lysosomal compartments, and liposome assays showed that SAP-C efficiently extracts antigen from membranes. Coprecipitation demonstrated direct molecular interaction between SAP-C and CD1b. We propose a model in which SAP-C exposes lipid antigens from intralysosomal membranes for loading onto CD1b. Thus, SAP-C represents a missing link in antigen presentation of lipids through CD1b to human T cells.


Subject(s)
Antigen Presentation , Antigens, CD1/metabolism , Glycoproteins/immunology , Lipids/immunology , T-Lymphocytes/immunology , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Fibroblasts/immunology , Fibroblasts/metabolism , Glycoproteins/deficiency , Glycoproteins/genetics , Humans , In Vitro Techniques , Lymphocyte Activation , Lysosomes/immunology , Lysosomes/metabolism , Protein Binding , Saposins , Sphingolipid Activator Proteins , T-Lymphocytes/metabolism , Transfection , beta-Glucosidase/metabolism
9.
Biochimie ; 85(3-4): 439-48, 2003.
Article in English | MEDLINE | ID: mdl-12770782

ABSTRACT

The physiological degradation of several membrane-bound glycosphingolipids (GSLs) by water-soluble lysosomal exohydrolases requires the assistance of sphingolipid activator proteins (SAPs). Four of these SAPs are synthesized from a single precursor protein (prosaposin). Inherited deficiency of this precursor results in a rare disease in humans with an accumulation of ceramide (Cer) and glycolipids such as glucosylceramide and lactosylceramide (LacCer). In a previous study, we have shown that human SAP-D stimulates the lysosomal degradation of Cer in precursor deficient cells. In order to study the role of SAPs (or saposins) A-D in cellular GSL catabolism, we recently investigated the catabolism of exogenously added [(3)H]labeled ganglioside GM1, Forssman lipid, and endogenously [(14)C]labeled GSLs in SAP-precursor deficient human fibroblasts after the addition of recombinant SAP-A, -B, -C and -D. We found that activator protein deficient cells are still able to slowly degrade gangliosides GM1 and GM3, Forssman lipid and globotriaosylceramide to a significant extent, while LacCer catabolism critically depends on the presence of SAPs. The addition of either of the SAPs, SAP-A, SAP-B or SAP-C, resulted in an efficient hydrolysis of LacCer.


Subject(s)
Glycoproteins/metabolism , Glycosphingolipids/metabolism , Base Sequence , Cells, Cultured , Endocytosis , Fibroblasts/metabolism , G(M1) Ganglioside/metabolism , G(M3) Ganglioside/metabolism , Globosides/metabolism , Glycoproteins/deficiency , Glycosphingolipids/chemistry , Humans , Molecular Sequence Data , Saposins , Sphingolipid Activator Proteins , Trihexosylceramides/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...