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

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

doi:10.1074/jbc.m607281200

Cited by 72 publications

(60 citation statements)

References 55 publications

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“…8 ). These fi ndings confi rm earlier observations on the essential functions of Sap A and Sap B, for the glycosphingolipid metabolism, that are impaired by high cholesterol levels ( 7,8 ). Furthermore, it was shown that a defect of the protein NPC2 in Niemann-Pick disease type C not only caused an accumulation of cholesterol in the liver and brain, but also, as a secondary effect, caused increased storage of gangliosides such as GM2 ( 74,75 ).…”

Section: The His 6 -Tag Changes the Overall Properties Of Gm2apsupporting

confidence: 88%

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Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity

Anheuser

Breiden

Schwarzmann

et al. 2015

Journal of Lipid Research

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For the effective degradation of lysosomal sphingolipids with short oligosaccharide head groups, special lipid binding and transfer proteins (the sphingolipid activator proteins) are needed. The sphingolipid activator proteins consist of the GM2 activator protein (GM2AP) and the saposins (Saps) A-D. They all facilitate the interaction of the membrane-bound substrate with the water-soluble enzyme at the water-membrane interface and differ from sphingolipid transfer proteins of the cytosol ( 6 ). The anionic lysosomal phospholipid, BMP, is also known to dramatically enhance (glyco)sphingolipid hydrolysis by lysosomal hydrolases and sphingolipid activator proteins ( 7-9 ).Even at low pH values (pH <5.0), BMP conveys negative surface charge to luminal lysosomal vesicles ( 10,11 ). This favors an electrostatic binding of the positively charged activator proteins and the polycationic hydrolases, and enhances degradation of (glyco)sphingolipids substantially ( 7,9,10,(12)(13)(14)(15)(16).The GM2AP, which has a hydrophobic binding cavity for lipids and a binding site for ␤ -hexosaminidase A (HexA) ( 17-19 ), arranges the interaction of ganglioside GM2 with HexA and facilitates the formation of a Michaelis-Menten complex with access of the catalytic site to the carbohydrate head group ( 20,21 ). The whole working mechanism, Abstract Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with ␤ -hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. Components of eukaryotic plasma membranes reach the intraendolysosomal vesicles by endocytotic membrane fl ow ( 1 ). Those vesicles have been identifi ed as platforms for lipid and membrane degradation ( 2 ). They differ from the limiting endosomal membrane by the absence of a glycocalix, a lower cholesterol content, and the cumulative occurrence of the anionic bis(monoacylglycero)phosphate (BMP) ...

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Section: The His 6 -Tag Changes the Overall Properties Of Gm2apsupporting

confidence: 88%

“…This favors an electrostatic binding of the positively charged activator proteins and the polycationic hydrolases, and enhances degradation of (glyco)sphingolipids substantially ( 7,9,10,(12)(13)(14)(15)(16).…”

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confidence: 99%

Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity

Anheuser

Breiden

Schwarzmann

et al. 2015

Journal of Lipid Research

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“…Two recent reports have shown that glycosylated recombinant saposins A (37) and B (38) expressed in yeast differ from the unglycosylated forms in their ability to solubilize lipids from immobilized liposomes. However, earlier reports have shown that nonglycosylated saposins retain their lipid-binding and enzyme activation effects (39), and recombinant sapC expressed in Escherichia coli was shown to have similar properties to sapC purified from natural tissues (40).…”

Section: Discussionmentioning

confidence: 99%

Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

Alattia

Shaw²,

Yip³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Acid ␤-glucosidase (GCase) is a soluble lysosomal enzyme responsible for the hydrolysis of glucose from glucosylceramide and requires activation by the small nonenzymatic protein saposin C (sapC) to gain access to the membrane-embedded glycosphingolipid substrate. We have used in situ atomic force microscopy (AFM) with simultaneous confocal and epifluorescence microscopies to investigate the interactions of GCase and sapC with lipid bilayers. GCase binds to sites on membranes transformed by sapC, and enzyme activity occurs at loci containing both GCase and sapC. Using FRET, we establish the presence of GCase/sapC and GCase/ product contacts in the bilayer. These data support a mechanism in which sapC locally alters regions of bilayer for subsequent attack by the enzyme in stably bound protein complexes.atomic force microscopy ͉ confocal microscopy ͉ FRET ͉ interfacial catalysis ͉ lipid storage disease

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“…To investigate the role of saposins as lipid transfer proteins independently of any storage phenotype, we designed cell-free assays and measured binding of Escherichia coli-purified recombinant saposins to synthetic lipids and subsequent loading of CD1d molecules. Although there is evidence that glycosylation influences the ability of recombinant saposins to mobilize lipids from membranes and liposomes (51,52), nonglycosylated proteins have been shown to retain comparable lipid binding and hydrolase-activating capacity. Despite high homology, the four saposins have different lipid specificities, as demonstrated by the phenotype of patients with selective deficiency of individual molecules (53).…”

Section: Discussionmentioning

confidence: 99%

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

Salio

Ghadbane

Dushek³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Lipid transfer proteins, such as molecules of the saposin family, facilitate extraction of lipids from biological membranes for their loading onto CD1d molecules. Although it has been shown that prosaposin-deficient mice fail to positively select invariant natural killer T (iNKT) cells, it remains unclear whether saposins can facilitate loading of endogenous iNKT cell agonists in the periphery during inflammatory responses. In addition, it is unclear whether saposins, in addition to loading, also promote dissociation of lipids bound to CD1d molecules. To address these questions, we used a combination of cellular assays and demonstrated that saposins influence CD1d-restricted presentation to human iNKT cells not only of exogenous lipids but also of endogenous ligands, such as the self-glycosphingolipid β-glucopyranosylceramide, up-regulated by antigen-presenting cells following bacterial infection. Furthermore, we demonstrated that in human myeloid cells CD1d-loading of endogenous lipids after bacterial infection, but not at steady state, requires trafficking of CD1d molecules through an endo-lysosomal compartment. Finally, using BIAcore assays we demonstrated that lipid-loaded saposin B increases the offrate of lipids bound to CD1d molecules, providing important insights into the mechanisms by which it acts as a "lipid editor," capable of fine-tuning loading and unloading of CD1d molecules. These results have important implications in understanding how to optimize lipid-loading onto antigen-presenting cells, to better harness iNKT cells central role at the interface between innate and adaptive immunity. autoreactivity | inflammation | innate immunity | lipid binding proteins

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

Cited by 72 publications

References 55 publications

Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity

Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity

Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

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