Degradation of Membrane-bound Ganglioside GM1

Wilkening, Gundo; Linke, Thomas; Uhlhorn-Dierks, Gunther; Sandhoff, Konrad

doi:10.1074/jbc.m006568200

Cited by 80 publications

(43 citation statements)

References 24 publications

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“…Similar results were obtained for the degradation of glucosylceramide by glucocerebrosidase and SAP-C (4) and of GM1 by ␤-galactosidase and SAP-B or GM2AP (35). These results together with the data presented here strongly support our hypothesis that the lysosomal degradation of glycosphingolipids takes place on intralysosomal membrane structures rather than in the limiting lysosomal membrane.…”

Section: Discussionsupporting

confidence: 81%

“…This demonstrates that GM2AP shows some specificity but is not completely specific for GM2, which is in agreement with earlier glycolipid transfer studies using GM2AP (5,28). An interesting point is that studies on the enzymatic degradation of GM1 led to the conclusion that the GM2AP, similar to SAP-B, stimulates this degradation step in the presence of BMP (35). Whether, therefore, the strong drop in the plasmon resonance signal, which was observed for GM1 and GM2, is due to the same effect that causes the activator function of GM2AP is still speculative, but an interesting point to consider.…”

Section: Discussionsupporting

confidence: 77%

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Degradation of Membrane-bound Ganglioside GM2 by β-Hexosaminidase A

Werth

Schuette

Wilkening

et al. 2001

Journal of Biological Chemistry

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According to a recent hypothesis, glycosphingolipids originating from the plasma membrane are degraded in the acidic compartments of the cell as components of intraendosomal and intralysosomal vesicles and structures. Since most previous in vitro investigations used micellar ganglioside GM2 as substrate, we studied the degradation of membrane-bound ganglioside GM2 by water-soluble ␤-hexosaminidase A in the presence of the GM2 activator protein in a detergent-free, liposomal assay system. Our results show that anionic lipids such as the lysosomal components bis(monoacylglycero)phosphate or phosphatidylinositol stimulate the degradation of GM2 by ␤-hexosaminidase A up to 180-fold in the presence of GM2 activator protein. In contrast, the degradation rate of GM2 incorporated into liposomes composed of neutral lysosomal lipids such as dolichol, cholesterol, or phosphatidylcholine was significantly lower than in negatively charged liposomes. This demonstrates that both, the GM2 activator protein and anionic lysosomal phospholipids, are needed to achieve a significant degradation of membrane-bound GM2 under physiological conditions. The interaction of GM2 activator protein with immobilized membranes was studied with surface plasmon resonance spectroscopy at an acidic pH value as it occurs in the lysosomes. Increasing the concentration of bis(monoacylglycero)phosphate in immobilized liposomes led to a significant drop of the resonance signal in the presence of GM2 activator protein. This suggests that in the presence of bis(monoacylglycero)phosphate, which has been shown to occur in inner membranes of the acidic compartment, GM2 activator protein is able to solubilize lipids from the surface of immobilized membrane structures.The degradation of glycosphingolipids (GSLs) 1 endocytosed from the plasma membrane takes place in the acidic compartments of the cell. According to a recently proposed hypothesis of the topology of lysosomal digestion (1), GSLs reach the lysosomal compartments as membrane-bound components of intraendosomal and intralysosomal vesicles and structures. The degradation of GSLs with short oligosaccharide head groups and ceramide requires two proteins: a water-soluble lysosomal sphingolipid hydrolase and a sphingolipid activator protein (SAP) (2, 3). Recent studies suggest that SAPs mediate the interaction of water-soluble sphingolipid hydrolases with their respective membrane-bound substrates (4). The enzymatic, lysosomal degradation of the ganglioside GM2 is catalyzed by ␤-hexosaminidase A (HexA) and requires the GM2 activator protein (GM2AP) as cofactor (5).Human lysosomal ␤-hexosaminidases (EC 3.2.1.52) cleave off terminal ␤-glycosidically-bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates, including glycoproteins, oligosaccharides, and GSLs such as GM2, its asialo derivative GA2, and globoside (6). A total of three different ␤-hexosaminidase isoenzymes have been identified. These are composed of different combinations of two noncovalently linked, structur...

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Section: Discussionsupporting

confidence: 81%

Section: Discussionsupporting

confidence: 77%

Degradation of Membrane-bound Ganglioside GM2 by β-Hexosaminidase A

Werth

Schuette

Wilkening

et al. 2001

Journal of Biological Chemistry

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“…1 that lysosomal anionic lipids like BMP and PI stimulate the hydrolysis of ceramide. Both lipids are negatively charged under the chosen assay conditions (27,28). The pI value of human AC was calculated to be 7.2, and AC is therefore likely to be positively charged in a lysosomal environment.…”

Section: Anionic Phospholipids and Lysosomal Degradation-accord-mentioning

confidence: 99%

Interfacial Regulation of Acid Ceramidase Activity

Linke¹,

Wilkening²,

Sadeghlar³

et al. 2001

Journal of Biological Chemistry

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110

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The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis (monoacylglycero)

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“…Thus, saposin B is able to extract and solubilize cerebroside sulfates from membranes, allowing arylsulfatase A to act on the small, diffusible protein-lipid complexes. Saposin B may also have a physiological role in activating the hydrolysis of the ganglioside GM1 to GM2 by lysosomal ␤-galactosidase, and in this case, the activator may act by modifying the local lipid structure at the membrane surface to allow catalysis to proceed (4).…”

mentioning

confidence: 99%

Crystal structure of saposin B reveals a dimeric shell for lipid binding

Ahn

Faull²,

Whitelegge³

et al. 2002

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

174

224

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Saposin B is a small, nonenzymatic glycosphingolipid activator protein required for the breakdown of cerebroside sulfates (sulfatides) within the lysosome. The protein can extract target lipids from membranes, forming soluble protein-lipid complexes that are recognized by arylsulfatase A. The crystal structure of human saposin B reveals an unusual shell-like dimer consisting of a monolayer of ␣-helices enclosing a large hydrophobic cavity. Although the secondary structure of saposin B is similar to that of the known monomeric members of the saposin-like superfamily, the helices are repacked into a different tertiary arrangement to form the homodimer. A comparison of the two forms of the saposin B dimer suggests that extraction of target lipids from membranes involves a conformational change that facilitates access to the inner cavity.T he saposins are a group of four structurally related activator proteins that function in conjunction with hydrolase enzymes for the stepwise breakdown of glycosphingolipids within the lysosome (1, 2). These proteins act by modifying the local environment of target lipids, and ''activate'' the breakdown of their substrates by presenting them in a form in which the lipid scissile bonds are accessible to the active sites of the specific enzymes. Presumably, the hydrolases cannot form a catalytic complex with the glycosphingolipids when these are in unmodified membrane bilayers.Each of the four saposins activates one or more lipid exohydrolases. For example, saposin B (also known as the cerebroside sulfate activator, or CS-Act) facilitates the hydrolysis of the sulfate group from cerebroside sulfate by arylsulfatase A, resulting in the formation of galactosylceramide (3). This glycolipid is then catabolized to ceramide by ␤-galactosylceramidase in a reaction activated by saposin C. There seems to be more than one mechanism of activation by the saposins, and this may further depend on the particular target lipid. Thus, saposin B is able to extract and solubilize cerebroside sulfates from membranes, allowing arylsulfatase A to act on the small, diffusible protein-lipid complexes. Saposin B may also have a physiological role in activating the hydrolysis of the ganglioside GM1 to GM2 by lysosomal ␤-galactosidase, and in this case, the activator may act by modifying the local lipid structure at the membrane surface to allow catalysis to proceed (4).The saposins belong to a large and diverse family of small, cysteine-rich proteins that share a common ability to interact with membranes but act in a wide variety of functions. Other members of the ''saposin-like'' superfamily of proteins include the lung surfactant-associated protein B (SP-B), the tumorolytic protein NK-lysin, granulysin, the pore-forming amoebapores, and the membrane-targeting domain of some enzymes (5, 6). The saposin-like proteins are Ϸ80 residues in length and have a characteristic pattern of conserved cysteines ( Fig. 1A; refs. 7 and 8). To date, the three-dimensional modeling of the saposins and other members of the sapo...

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Degradation of Membrane-bound Ganglioside GM1

Cited by 80 publications

References 24 publications

Degradation of Membrane-bound Ganglioside GM2 by β-Hexosaminidase A

Degradation of Membrane-bound Ganglioside GM2 by β-Hexosaminidase A

Interfacial Regulation of Acid Ceramidase Activity

Crystal structure of saposin B reveals a dimeric shell for lipid binding

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