Complete localization of disulfide bonds in GM2 activator protein

Schutte, Chantelle; Lemm, Thorsten; Glombitza, Gereon J.; Sandhoff, Konrad

doi:10.1002/pro.5560070421

Cited by 32 publications

(14 citation statements)

References 22 publications

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“…This forms four disulfide bridges whose mode of coupling was recently elucidated by the combination of protein-chemical and mass spectrometric techniques. [281] The cDNA and large regions of the gene are known. [282] Mutations in AB-patients have been identified.…”

Section: Ab-variant Of Gm2-gangliosidosesmentioning

confidence: 99%

Sphingolipids—Their Metabolic Pathways and the Pathobiochemistry of Neurodegenerative Diseases

Kolter

Sandhoff

1999

Angew. Chem. Int. Ed.

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385

268

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Section: Ab-variant Of Gm2-gangliosidosesmentioning

confidence: 99%

Sphingolipids—Their Metabolic Pathways and the Pathobiochemistry of Neurodegenerative Diseases

Kolter

Sandhoff

1999

Angew. Chem. Int. Ed.

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385

268

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“…Recombinant GM2AP was expressed in insect cells using the baculovirus expression system and purified as described previously (16).…”

Section: Activator Preparationmentioning

confidence: 99%

Physiological Substrates for Human Lysosomal β-Hexosaminidase S

Hepbildikler¹,

Sandhoff²,

Kölzer³

et al. 2002

Journal of Biological Chemistry

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Human lysosomal ␤-hexosaminidases remove terminal ␤-glycosidically bound N-acetylhexosamine residues from a number of glycoconjugates. Three different isozymes composed of two noncovalently linked subunits ␣ and ␤ exist: Hex A (␣␤), Hex B (␤␤), and Hex S (␣␣). While the role of Hex A and B for the degradation of several anionic and neutral glycoconjugates has been well established, the physiological significance of labile Hex S has remained unclear. However, the striking accumulation of anionic oligosaccharides in double knockout mice totally deficient in hexosaminidase activity but not in mice expressing Hex S (Sango, K., McDonald, M. P., Crawley, J. N., Mack, M. L., Tifft, C.J., Skop, E., Starr, C. M., Hoffmann, A., Sandhoff, K., Suzuki, K., and Proia, R. L., (1996) Nat. Genet. 14, 348 -352) prompted us to reinvestigate the substrate specificity of Hex S. To identify physiological substrates of Hex S, anionic and neutral oligosaccharides excreted in the urine of the double knockout mice were isolated and analyzed. Using ESI-MS/MS and glycosidase digestion the anionic glycans were identified as products of incomplete dermatan sulfate degradation whereas the neutral storage oligosaccharides were found to be fragments of N-glycan degradation. In vitro, recombinant Hex S was highly active on water-soluble and amphiphilic glycoconjugates including artificial substrates, sulfated GAG fragments, and the sulfated glycosphingolipid SM2. Hydrolysis of membrane-bound SM2 by the recombinant Hex S was synergistically stimulated by the GM2 activator protein and the lysosomal anionic phospholipid bis(monoacylglycero)phosphate.Lysosomal hexosaminidases (EC 3.2.1.52) release terminal ␤-glycosidically linked N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates (1). They are composed of two subunits, ␣ and ␤, derived from homologous genes HEXA and HEXB. Hexosaminidase A (Hex A, 1 ␣␤)and Hex B (␤␤) were believed to be the major functional isozymes, and Hex S (␣␣) a minor labile form without significant activity (2). Several attempts to isolate pure Hex S from human tissues or cell homogenates yielded preparations with poor enzymatic activity (3-5).Each subunit possesses an active site characterized by its own substrate specificity (5). The active site of the ␤-subunit hydrolyzes uncharged substrates, whereas the ␣-subunit, in addition, cleaves negatively charged substrates. Only the ␣␤-heterodimer Hex A is able to degrade ganglioside GM2 (Fig. 1B) at significant rates in the presence of the GM2 activator protein (GM2AP).A group of severe neurodegenerative storage diseases, the GM2 gangliosidoses, results from mutations in any of the genes encoding the two hexosaminidase subunits and GM2AP. TaySachs disease is caused by mutations in the HEXA gene resulting in a deficiency in Hex A and Hex S, whereas in Sandhoff disease the lack of Hex A and Hex B activity is observed due to mutations in the HEXB gene. The GM2 gangliosidoses are characterized by a massive accumulation of ganglioside GM2 and relat...

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“…Expression of the GM2 Activator Protein-The human GM2 activator protein (GM2-AP) was expressed in insect cells using the baculovirus expression system as described previously (19).…”

Section: Synthesis Of [mentioning

confidence: 99%

Degradation of Membrane-bound Ganglioside GM1

Wilkening¹,

Linke²,

Uhlhorn-Dierks³

et al. 2000

Journal of Biological Chemistry

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In this study we demonstrate that an activator protein is required for the enzymatic degradation of membrane-bound ganglioside GM1 and that both SAP-B and the GM2 activator protein significantly enhance the degradation of the ganglioside GM1 by acid ␤-galactosidase in a liposomal, detergent-free assay system. These findings offer a possible explanation for the observation that no storage of the ganglioside GM1 has been observed in patients with either isolated prosaposin or isolated GM2 activator deficiency. We also demonstrate that anionic phospholipids such as bis(monoacylglycero)phosphate and phosphatidylinositol, which specifically occur in inner membranes of endosomes and in lysosomes, are essential for the activator-stimulated hydrolysis of the ganglioside GM1. Assays utilizing surface plasmon resonance spectroscopy showed that bis(monoacylglycero)phosphate increases the binding of both ␤-galactosidase and activator proteins to substrate-carrying membranes.

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Complete localization of disulfide bonds in GM2 activator protein

Cited by 32 publications

References 22 publications

Sphingolipids—Their Metabolic Pathways and the Pathobiochemistry of Neurodegenerative Diseases

Sphingolipids—Their Metabolic Pathways and the Pathobiochemistry of Neurodegenerative Diseases

Physiological Substrates for Human Lysosomal β-Hexosaminidase S

Degradation of Membrane-bound Ganglioside GM1

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