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Konrad Sandhoff - One of the best experts on this subject based on the ideXlab platform.

  • Purification of acid ceramidase from human placenta.
    Methods in Enzymology, 2020
    Co-Authors: Thomas Linke, S. Lansmann, Konrad Sandhoff
    Abstract:

    Publisher Summary Human acid ceramidase (haCerase, EC 3.5.1.23, N-acylsphingosine amidohydrolase) is a lysosomal enzyme that catalyzes the hydrolysis of ceramide into sphingosine and free fatty acid. In vivo and in vitro studies on Sphingolipid metabolism showed that degradation of ceramide by haCerase is stimulated by a nonenzymic glycoProtein called “Sphingolipid Activator Protein D” (SAP-D). A deficiency in haCerase activity leads to the autosomal recessive Sphingolipid storage disorder called “Farber disease,” caused by accumulation of ceramide in lysosomes. Seven different phenotypes of this rare disorder are known to date. A purification procedure using human urine from patients with peritonitis as an enzyme source yielding an apparently homogeneous enzyme preparation and providing peptide sequence data that subsequently led to the isolation and characterization of a full-length cDNA encoding haCerase has been described. Because copurification of Sphingolipid Activator Protein C (SAP-C) and glucocerebrosidase is reported by Aerts et al., this chapter tests each purification step for the presence of SAP-D in haCerase-containing fractions. Western blot analysis indicates that SAP-D copurified with haCerase. To allow further functional and structural studies on haCerase, a purification procedure yielding SAP-D free, enzymatically active haCerase was established and optimized.

  • Mechanism of Secondary Ganglioside and Lipid Accumulation in Lysosomal Disease.
    International Journal of Molecular Sciences, 2020
    Co-Authors: Bernadette Breiden, Konrad Sandhoff
    Abstract:

    Gangliosidoses are caused by monogenic defects of a specific hydrolase or an ancillary Sphingolipid Activator Protein essential for a specific step in the catabolism of gangliosides. Such defects in lysosomal function cause a primary accumulation of multiple undegradable gangliosides and glycoSphingolipids. In reality, however, predominantly small gangliosides also accumulate in many lysosomal diseases as secondary storage material without any known defect in their catabolic pathway. In recent reconstitution experiments, we identified primary storage materials like sphingomyelin, cholesterol, lysoSphingolipids, and chondroitin sulfate as strong inhibitors of Sphingolipid Activator Proteins (like GM2 Activator Protein, saposin A and B), essential for the catabolism of many gangliosides and glycoSphingolipids, as well as inhibitors of specific catabolic steps in lysosomal ganglioside catabolism and cholesterol turnover. In particular, they trigger a secondary accumulation of ganglioside GM2, glucosylceramide and cholesterol in Niemann–Pick disease type A and B, and of GM2 and glucosylceramide in Niemann–Pick disease type C. Chondroitin sulfate effectively inhibits GM2 catabolism in mucopolysaccharidoses like Hurler, Hunter, Sanfilippo, and Sly syndrome and causes a secondary neuronal ganglioside GM2 accumulation, triggering neurodegeneration. Secondary ganglioside and lipid accumulation is furthermore known in many more lysosomal storage diseases, so far without known molecular basis.

  • functional characterization of the postulated intramolecular Sphingolipid Activator Protein domain of human acid sphingomyelinase
    Biological Chemistry, 2004
    Co-Authors: Melanie Kolzer, O. Bartelsen, Klaus Ferlinz, Silvia Locatelli Hoops, Florian Lang, Konrad Sandhoff
    Abstract:

    : Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of Sphingolipid Activator Proteins (Saps: saposins A-D; GM2 Activator) is not essential to mediate this reaction at the water-lipid interface in vivo . A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the Sphingolipid Activator Protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C.

  • Stimulation of acid sphingomyelinase activity by lysosomal lipids and Sphingolipid Activator Proteins
    Biological Chemistry, 2001
    Co-Authors: Thomas Linke, S. Lansmann, H. Moczall, O. Bartelsen, J. Weisgerber, G. Wilkening, Konrad Sandhoff
    Abstract:

    Acid sphingomyelinase is a water-soluble, lysosomal glycoProtein that catalyzes the degradation of mem- brane-bound sphingomyelin into phosphorylcholine and ceramide. Sphingomyelin itself is an important component of the extracellular leaflet of various cellu- lar membranes. The aim of the present investigation was to study sphingomyelin hydrolysis as a mem- brane-bound process. We analyzed the degradation of sphingomyelin by recombinant, highly purified acid sphingomyelinase in a detergent-free, liposomal as- say system. In order to mimic the in vivo intralysoso- mal conditions as closely as possible a number of negatively charged, lysosomally occuring lipids in- cluding bis(monoacylglycero)phosphate and phos- phatidylinositol were incorporated into substrate- carrying liposomes. Dolichol and its phosphate ester dolicholphosphate were also included in this study. Bis(monoacylglycero)phosphate and phosphatidyl- inositol were both effective stimulators of sphin- gomyelin hydrolysis. Dolichol and dolicholphosphate also significantly increased sphingomyelin hydrolysis. The influence of membrane curvature was investigat- ed by incorporating the substrate into small (SUVs) and large unilamellar vesicles (LUVs) with varying mean diameter. Degradation rates were substantially higher in SUVs than in LUVs. Surface plasmon reso- nance experiments demonstrated that acid sphin- gomyelinase binds strongly to lipid bilayers. This in- teraction is significantly enhanced by anionic lipids such as bis(monoacylglycero)phosphate. Under de- tergent-free conditions only the Sphingolipid Activator Protein SAP-C had a pronounced influence on sphin- gomyelin degradation in both neutral and negatively charged liposomes, catalyzed by highly purified acid sphingomyelinase, while SAP-A, -B and -D had no no- ticeable effect on sphingomyelin degradation.

  • quantification of mrnas encoding Proteins of the glycoSphingolipid catabolism in mouse models of gm2 gangliosidoses and Sphingolipid Activator Protein precursor prosaposin deficiency
    Biochimica et Biophysica Acta, 2000
    Co-Authors: Astrid Potratz, Silke Huttler, Uwe Bierfreund, Richard L Proia, Kunihiko Suzuki, Konrad Sandhoff
    Abstract:

    Abstract We have investigated the mRNA amounts of six lysosomal Proteins (β-hexosaminidase α- and β-subunit, Sphingolipid Activator Protein precursor, GM2 Activator Protein, lysosomal sialidase, β-glucocerebrosidase) involved in the degradation of glycoSphingolipids. We analyzed extracts from brain tissues of mouse models for lysosomal storage diseases, i.e., the GM2 gangliosidoses and the deficiency of the Sphingolipid Activator Protein precursor (prosaposin). The mRNA levels were quantified by real-time reverse transcription–polymerase chain reaction. Although storage of the respective lysosomal Proteins has been reported in human and mice, no increase of their mRNA amounts could be detected here. Our results indicate that there is no transcriptional upregulation of lysosomal Proteins in the examined neuronal storage disorders.

Klaus Harzer - One of the best experts on this subject based on the ideXlab platform.

  • Sphingolipid Activator Protein d sap d stimulates the lysosomal degradation of ceramide in vivo
    Biochemical and Biophysical Research Communications, 1994
    Co-Authors: Andreas Klein, Kunihiko Suzuki, Klaus Harzer, Margarete Henseler, C Klein, Konrad Sandhoff
    Abstract:

    Abstract GlycoSphingolipids of cultured fibroblasts from patients with total Sphingolipid Activator Proteins (SAPs) deficiency (Schnabel et al. J. Biol. Chem. 267:3312-3315, 1992) were labeled biosynthetically with [14C]serine. After a chase period of 120h the patients′ fibroblasts showed increased labeling of ceramide, glucosylceramide, lactosylceramide and ganglioside GM3 in comparison to normal control cells. Addition of sap-D to the chase-media of the patients′ fibroblasts led to the degradation of the accumulated ceramide down to nearly normal levels, whereas the levels of other labeled Sphingolipids remained unaffected. In contrast, addition of sap-B to the chase-media of the patients′ fibroblasts did not reduce the increased ceramide levels but resulted, as expected from in vitro experiments, in a specific decrease of levels of lactosylceramide and ganglioside GM3 Therefore,we conclude that sap-D stimulates in vivo the lysosomal degradation of ceramide.

  • prosaposin deficiency further characterization of the Sphingolipid Activator Protein deficient sibs multiple glycolipid elevations including lactosylceramidosis partial enzyme deficiencies and ultrastructure of the skin in this generalized sphingolip
    Human Genetics, 1993
    Co-Authors: V. Bradová, Barbara C Paton, F. Šmíd, W. Roggendorf, B Ulrichbott, Klaus Harzer
    Abstract:

    Sphingolipid Activator Protein (SAP) deficiency, previously described in two sibs and shown to be caused by the absence of the common saposin precursor (prosaposin), was further characterized by biochemical lipid and enzyme studies and by ultrastructural analysis. The 20 week old fetal sib had increased concentrations of neutral glycolipids, including mono-, di-, tri- and tetrahexosylceramide, in liver, kidney and cultured skin fibroblasts compared with the controls. Glucosylceramide and lactosylceramide were particularly elevated. The kidney of the affected fetus showed additional increases in the concentration of sulphatide, galactosylceramide and digalactosylceramide. Free ceramide was stored in the liver and kidney, and GM3 and GM2 gangliosides were elevated in the liver, but not the brain, of the fetus. Phospholipids, however, were normal in the affected fetus. In the liver biopsy of the propositus, who later died at 16 weeks of age, only a few lipids could be studied. Glucosylceramide, dihexosylceramide and ceramide were elevated in agreement with our previous study. Enzyme studies were undertaken using detergent free liposomal substrate preparations and fibroblast extracts. The sibs' β-glucocerebrosidase and β-galactocerebrosidase activities were clearly reduced, but their sphingomyelinase activities were normal. The normal activity of the latter enzyme and the almost normal tissue concentration of sphingomyelin in prosaposin deficiency suggest that the prosaposin derived SAPs are not required for sphingomyelinase activity in vivo. In keeping with the biochemical findings, skin biopsies from the sibs showed massive lysosomal storage with a vesicular and membranous ultrastructure. The function of SAPs in Sphingolipid degradation and the role of SAPs for enzyme activity in vitro are discussed. In addition, the similarity in neutral glycolipid accumulations in Niemann Pick disease type C and in prosaposin deficiency are noted. The phenotype of the prosaposin deficient sibs resembled acute neuronopathic (type 2) Gaucher disease more than Farber disease in several aspects, but their genotype was unique.

  • additional biochemical findings in a patient and fetal sibling with a genetic defect in the Sphingolipid Activator Protein sap precursor prosaposin evidence for a deficiency in sap 1 and for a normal lysosomal neuraminidase
    Biochemical Journal, 1992
    Co-Authors: Barbara C Paton, A Poulos, B Kustermannkuhn, B Schmid, Klaus Harzer
    Abstract:

    It has been shown that Sphingolipid Activator Proteins (SAPs) 1 and 2 are encoded on the same gene along with two other putative Activator Proteins [Furst, Machleidt & Sandhoff (1988) Biol. Chem. Hoppe-Seyler 369, 317-328 and O'Brien, Kretz, Dewji, Wenger, Esch & Fluharty (1988) Science 241, 1098-1101]. We have undertaken further biochemical investigations on a patient and fetal sibling, who were previously shown to have a unique Sphingolipid storage disorder associated with an SAP-2 deficiency [Harzer, Paton, Poulos, Kustermann-Kuhn, Roggendorf, Grisar & Popp (1989) Eur. J. Pediatr. 149, 31-39]. The severity of their disorder suggested that other products of the SAP precursor or prosaposin gene may also be deficient. The turnover of cerebroside sulphate and globotriaosylceramide were investigated and were both impaired in fibroblasts from the patient and fetus. However, the activities of cerebroside sulphate sulphatase and globotriaosylceramide alpha-galactosidase in vitro were normal in cells from the fetus and patient respectively. In addition, there was an increase in cerebroside sulphate concentration in the kidney of the affected fetus. These results indicate that, in addition to the SAP-2 deficiency, there was a defect in SAP-1 function in this disorder. Additional increases in the concentration of monohexosyl- and dihexosyl-ceramide in the fetal kidney probably reflect the deficiency of SAP-2 in the case of monohexosylceramides, and the combined Activator deficiency in the case of dihexosylceramides. Lactosylceramide-loading studies confirmed that there was a defect in the turnover of this lipid in fibroblasts from the affected patient and fetus but not from a patient with an isolated SAP-1 deficiency, or from patients with Krabbe disease, GM1 gangliosidosis or galactosialidosis. It has been suggested [Potier, Lamontagne, Michaud & Tranchemontagne (1990) Biochem. Biophys. Res. Commun. 173, 449-456] that the prosaposin gene also codes for lysosomal neuroaminidase. However, we found normal neuraminidase activity in fibroblasts from our patient, using assay conditions which are diagnostic for sialidosis patients. The role of prosaposin gene products in Sphingolipid metabolism is discussed in view of our biochemical findings in this genetic disorder.

  • metabolism of gm1 ganglioside in cultured skin fibroblasts anomalies in gangliosidoses sialidoses and Sphingolipid Activator Protein sap saposin 1 and prosaposin deficient disorders
    Human Genetics, 1992
    Co-Authors: B Schmid, Konrad Sandhoff, Barbara C Paton, Klaus Harzer
    Abstract:

    Cultured skin fibroblasts from controls and patients with lysosomal storage diseases were loaded with GM1 ganglioside that had been labelled with tritium in its ceramide moiety. After a 65-h or 240-h incubation, a large percentage of this ganglioside remained undegraded in GM1 gangliosidoses, whereas in the other storage diseases studied, one of its metabolites accumulated by 2–4 fold relative to controls. Labelled GM2 ganglioside accumulated in 4 variants of GM2 gangliosidosis, whereas labelled GM3 ganglioside accumulated in sialidosis, galactosialidoses and Sphingolipid Activator Protein 1 (SAP-1, saposin B) and prosaposin (saposin A, B, C an D) deficient lipidoses. The reduced degradation of GM3 ganglioside in the SAP-1 and prosaposin deficiencies was attributed to the deficient function of SAP-1. The prosaposin deficient cells also showed a reduced re-utilization of radioactive metabolites from GM1 ganglioside (i.e. sphingosine and fatty acid) for phospholipid biosynthesis compared with fibroblasts from the SAP-1 deficient patient or normal controls. This anomaly was ascribed to the previously shown defect in ceramide degradation in prosaposin deficiency.

  • Sphingolipid Activator Protein 1 deficiency in metachromatic leucodystrophy with normal arylsulphatase a activity a clinical morphological biochemical and immunological study
    European Journal of Pediatrics, 1991
    Co-Authors: W Schlote, Barbara C Paton, Klaus Harzer, H Christomanou, B Kustermannkuhn, B Schmid, J Seeger, U Beudt, I Schuster, U Langenbeck
    Abstract:

    A 7-year-old boy had clinical features of metachromatic leucodystrophy (MLD), however, an increased urinary sulphatide excretion was found in the presence of normal arylsulphatase A (and α-galactosidase A) activity. A rectal biopsy showed metachromatically staining storage macrophages as well as nonmetachromatic, but PAS-positive, submucosal neurons filled with membranous cytoplasmic bodies. These two types of storage material led to testing for a Sphingolipid Activator Protein (SAP) deficiency. Loading tests with sulphatide and globotriaosylceramide showed deficient turnover of both Sphingolipids in cultured fibroblasts. Using the Ouchterlony method, there was no reactivity between a described anti-SAP 1 antiserum and the patient's fibroblast extracts. This new case of SAP-1 deficient MLD was compared with the four cases of this variant known from the literature. Our results indicate that rectal biopsy morphology and lipid loading biochemistry should prove useful for the screening of SAP defects.

Kunihiko Suzuki - One of the best experts on this subject based on the ideXlab platform.

  • a mutation in the saposin c domain of the Sphingolipid Activator Protein prosaposin gene causes neurodegenerative disease in mice
    Journal of Neuroscience Research, 2010
    Co-Authors: Azusa Yoneshige, Kunihiko Suzuki, Kinuko Suzuki, Junko Matsuda
    Abstract:

    Saposins A, B, C, and D are small amphiphatic glycoProteins that are encoded in tandem within a precursor Protein (prosaposin, PSAP), and are required for in vivo degradation of Sphingolipids. Humans with saposin C deficiency exhibit the clinical presentation of Gaucher-like disease. We generated two types of saposin C mutant mice, one carrying a homozygous missense mutation (C384S) in the saposin C domain of prosaposin (Sap-C−/−) and the other carrying the compound heterozygous mutation with a second null Psap allele (Psap−/C384S). During early life stages, both Sap-C−/− and Psap−/C384S mice grew normally; however, they developed progressive motor and behavioral deficits after 3 months of age and the majority of affected mice could scarcely move by about 15 months. They showed no signs of hepatosplenomegaly throughout their lives. No accumulation of glucosylceramide and glucosylsphingosine was detected in the brain or liver of both Sap-C−/− and Psap−/C384S mice. Neuropathological analyses revealed patterned loss of cerebellar Purkinje cells, widespread axonal spheroids filled with membrane-derived concentric or lamellar electron-dense bodies, and lipofuscin-like deposition in the neurons. Soap-bubble-like inclusion bodies were detected in the trigeminal ganglion cells and the vascular endothelial cells. Compound heterozygous Psap−/C384S mice showed qualitatively identical but faster progression of the neurological phenotypes than Sap-C−/− mice. These results suggest the in vivo role of saposin C in axonal membrane homeostasis, the disruption of which leads to neurodegeneration in lysosomal storage disease. © 2010 Wiley-Liss, Inc.

  • the function of Sphingolipids in the nervous system lessons learnt from mouse models of specific Sphingolipid Activator Protein deficiencies
    Journal of Neurochemistry, 2007
    Co-Authors: Junko Matsuda, Azusa Yoneshige, Kunihiko Suzuki
    Abstract:

    We have generated specific saposin A and D deficient mouse mutants by the gene targeting technology. Saposin A deficient mice showed the clinical, biochemical and pathological phenotype of a chronic form of globoid cell leukodystrophy (Krabbe disease) establishing that saposin A is essential for in vivo degradation of galactosylceramide. Saposin D deficient mice showed an accumulation of ceramides containing α-hydroxy fatty acids (HFA/d18:1) in the brain and kidney and showed renal tubular degeneration and cerebellar Purkinje cell loss. Here we review the current information which we have learnt from these mouse models of specific Sphingolipid Activator Protein deficiencies. Collectively, the information provides support for the potential importance of Sphingolipids in the function of the nervous system.

  • mutation in saposin d domain of Sphingolipid Activator Protein gene causes urinary system defects and cerebellar purkinje cell degeneration with accumulation of hydroxy fatty acid containing ceramide in mouse
    Human Molecular Genetics, 2004
    Co-Authors: Junko Matsuda, Kunihiko Suzuki, Makiko Kido, Keiko Tadanoaritomi, Ineo Ishizuka, Kumiko Tominaga, Kazunori Toida, Eiji Takeda, Yasuhiro Kuroda
    Abstract:

    : The Sphingolipid Activator Proteins (saposins A, B, C and D) are small homologous glycoProteins that are encoded by a single gene in tandem within a large precursor Protein (prosaposin) and are required for in vivo degradation of some Sphingolipids with relatively short carbohydrate chains. Human patients with prosaposin or specific saposin B or C deficiency are known, and prosaposin- and saposin A-deficient mouse lines have been generated. Experimental evidence suggests that saposin D may be a lysosomal acid ceramidase Activator. However, no specific saposin D deficiency state is known in any mammalian species. We have generated a specific saposin D(-/-) mouse by introducing a mutation (C509S) into the saposin D domain of the mouse prosaposin gene. Saposin D(-/-) mice developed progressive polyuria at around 2 months and ataxia at around 4 months. Pathologically, the kidney of saposin D(-/-) mice showed renal tubular degeneration and eventual hydronephrosis. In the nervous system, progressive and selective loss of the cerebellar Purkinje cells in a striped pattern was conspicuous, and almost all Purkinje cells disappeared by 12 months. Biochemically, ceramides, particularly those containing hydroxy fatty acids accumulated in the kidney and the brain, most prominently in the cerebellum. These results not only indicate the role of saposin D in in vivo ceramide metabolism, but also suggest possible cytotoxicity of ceramide underlying the cerebellar Purkinje cell and renal tubular cell degeneration.

  • a mutation in the saposin a domain of the Sphingolipid Activator Protein prosaposin gene results in a late onset chronic form of globoid cell leukodystrophy in the mouse
    Human Molecular Genetics, 2001
    Co-Authors: Junko Matsuda, Marie T Vanier, Kinuko Suzuki, Jun Tohyama, Yuko Saito, Kunihiko Suzuki
    Abstract:

    : Sphingolipid Activator Proteins (saposins A, B, C and D) are small homologous glycoProteins derived from a common precursor Protein (prosaposin) encoded by a single gene. They are required for in vivo degradation of Sphingolipids with short carbohydrate chains. Six cysteines and one glycosylation site are strictly conserved in all four saposins. Total deficiency of all saposins and specific deficiency of saposin B or C are known among human patients. A mouse model of total saposin deficiency closely mimics the human disease. However, no specific saposin A or D deficiency is known. We introduced an amino acid substitution (C106F) into the saposin A domain by the Cre/loxP system which eliminated one of the three conserved disulfide bonds. Saposin A(-/-) mice developed slowly progressive hind leg paralysis with clinical onset at approximately 2.5 months and survival up to 5 months. Tremors and shaking, prominent in other myelin mutants, were not obvious until the terminal stage. Pathology and analytical biochemistry were qualitatively identical to, but generally much milder than, that seen in the typical infantile globoid cell leukodystrophy (GLD) in man (Krabbe disease) and in several other mammalian species, due to genetic deficiency of lysosomal galactosylceramidase (GALC) (EC 3.2.1.46). Thus, saposin A is indispensable for in vivo degradation of galactosylceramide by GALC. It should now be recognized that, in addition to GALC deficiency, genetic saposin A deficiency could also cause chronic GLD. Genetic saposin A deficiency might be anticipated among human patients with undiagnosed late-onset chronic leukodystrophy without GALC deficiency.

  • A mutation in the saposin A domain of the Sphingolipid Activator Protein (prosaposin) gene results in a late-onset, chronic form of globoid cell leukodystrophy in the mouse.
    Human Molecular Genetics, 2001
    Co-Authors: Junko Matsuda, Marie T Vanier, Kinuko Suzuki, Jun Tohyama, Yuko Saito, Kunihiko Suzuki
    Abstract:

    Sphingolipid Activator Proteins (saposins A, B, C and D) are small homologous glycoProteins derived from a common precursor Protein (prosaposin) encoded by a single gene. They are required for in vivo degradation of Sphingolipids with short carbohydrate chains. Six cysteines and one glycosylation site are strictly conserved in all four saposins. Total deficiency of all saposins and specific deficiency of saposin B or C are known among human patients. A mouse model of total saposin deficiency closely mimics the human disease. However, no specific saposin A or D deficiency is known. We introduced an amino acid substitution (C106F) into the saposin A domain by the Cre/loxP system which eliminated one of the three conserved disulfide bonds. Saposin A -/- mice developed slowly progressive hind leg paralysis with clinical onset at ∼2.5 months and survival up to 5 months. Tremors and shaking, prominent in other myelin mutants, were not obvious until the terminal stage. Pathology and analytical biochemistry were qualitatively identical to, but generally much milder than, that seen in the typical infantile globoid cell leukodystrophy (GLD) in man (Krabbe disease) and in several other mammalian species, due to genetic deficiency of lysosomal galactosylceramidase (GALC) (EC 3.2.1.46). Thus, saposin A is indispensable for in vivo degradation of galactosylceramide by GALC. It should now be recognized that, in addition to GALC deficiency, genetic saposin A deficiency could also cause chronic GLD. Genetic saposin A deficiency might be anticipated among human patients with undiagnosed late-onset chronic leukodystrophy without GALC deficiency.

Barbara C Paton - One of the best experts on this subject based on the ideXlab platform.

  • prosaposin deficiency further characterization of the Sphingolipid Activator Protein deficient sibs multiple glycolipid elevations including lactosylceramidosis partial enzyme deficiencies and ultrastructure of the skin in this generalized sphingolip
    Human Genetics, 1993
    Co-Authors: V. Bradová, Barbara C Paton, F. Šmíd, W. Roggendorf, B Ulrichbott, Klaus Harzer
    Abstract:

    Sphingolipid Activator Protein (SAP) deficiency, previously described in two sibs and shown to be caused by the absence of the common saposin precursor (prosaposin), was further characterized by biochemical lipid and enzyme studies and by ultrastructural analysis. The 20 week old fetal sib had increased concentrations of neutral glycolipids, including mono-, di-, tri- and tetrahexosylceramide, in liver, kidney and cultured skin fibroblasts compared with the controls. Glucosylceramide and lactosylceramide were particularly elevated. The kidney of the affected fetus showed additional increases in the concentration of sulphatide, galactosylceramide and digalactosylceramide. Free ceramide was stored in the liver and kidney, and GM3 and GM2 gangliosides were elevated in the liver, but not the brain, of the fetus. Phospholipids, however, were normal in the affected fetus. In the liver biopsy of the propositus, who later died at 16 weeks of age, only a few lipids could be studied. Glucosylceramide, dihexosylceramide and ceramide were elevated in agreement with our previous study. Enzyme studies were undertaken using detergent free liposomal substrate preparations and fibroblast extracts. The sibs' β-glucocerebrosidase and β-galactocerebrosidase activities were clearly reduced, but their sphingomyelinase activities were normal. The normal activity of the latter enzyme and the almost normal tissue concentration of sphingomyelin in prosaposin deficiency suggest that the prosaposin derived SAPs are not required for sphingomyelinase activity in vivo. In keeping with the biochemical findings, skin biopsies from the sibs showed massive lysosomal storage with a vesicular and membranous ultrastructure. The function of SAPs in Sphingolipid degradation and the role of SAPs for enzyme activity in vitro are discussed. In addition, the similarity in neutral glycolipid accumulations in Niemann Pick disease type C and in prosaposin deficiency are noted. The phenotype of the prosaposin deficient sibs resembled acute neuronopathic (type 2) Gaucher disease more than Farber disease in several aspects, but their genotype was unique.

  • additional biochemical findings in a patient and fetal sibling with a genetic defect in the Sphingolipid Activator Protein sap precursor prosaposin evidence for a deficiency in sap 1 and for a normal lysosomal neuraminidase
    Biochemical Journal, 1992
    Co-Authors: Barbara C Paton, A Poulos, B Kustermannkuhn, B Schmid, Klaus Harzer
    Abstract:

    It has been shown that Sphingolipid Activator Proteins (SAPs) 1 and 2 are encoded on the same gene along with two other putative Activator Proteins [Furst, Machleidt & Sandhoff (1988) Biol. Chem. Hoppe-Seyler 369, 317-328 and O'Brien, Kretz, Dewji, Wenger, Esch & Fluharty (1988) Science 241, 1098-1101]. We have undertaken further biochemical investigations on a patient and fetal sibling, who were previously shown to have a unique Sphingolipid storage disorder associated with an SAP-2 deficiency [Harzer, Paton, Poulos, Kustermann-Kuhn, Roggendorf, Grisar & Popp (1989) Eur. J. Pediatr. 149, 31-39]. The severity of their disorder suggested that other products of the SAP precursor or prosaposin gene may also be deficient. The turnover of cerebroside sulphate and globotriaosylceramide were investigated and were both impaired in fibroblasts from the patient and fetus. However, the activities of cerebroside sulphate sulphatase and globotriaosylceramide alpha-galactosidase in vitro were normal in cells from the fetus and patient respectively. In addition, there was an increase in cerebroside sulphate concentration in the kidney of the affected fetus. These results indicate that, in addition to the SAP-2 deficiency, there was a defect in SAP-1 function in this disorder. Additional increases in the concentration of monohexosyl- and dihexosyl-ceramide in the fetal kidney probably reflect the deficiency of SAP-2 in the case of monohexosylceramides, and the combined Activator deficiency in the case of dihexosylceramides. Lactosylceramide-loading studies confirmed that there was a defect in the turnover of this lipid in fibroblasts from the affected patient and fetus but not from a patient with an isolated SAP-1 deficiency, or from patients with Krabbe disease, GM1 gangliosidosis or galactosialidosis. It has been suggested [Potier, Lamontagne, Michaud & Tranchemontagne (1990) Biochem. Biophys. Res. Commun. 173, 449-456] that the prosaposin gene also codes for lysosomal neuroaminidase. However, we found normal neuraminidase activity in fibroblasts from our patient, using assay conditions which are diagnostic for sialidosis patients. The role of prosaposin gene products in Sphingolipid metabolism is discussed in view of our biochemical findings in this genetic disorder.

  • metabolism of gm1 ganglioside in cultured skin fibroblasts anomalies in gangliosidoses sialidoses and Sphingolipid Activator Protein sap saposin 1 and prosaposin deficient disorders
    Human Genetics, 1992
    Co-Authors: B Schmid, Konrad Sandhoff, Barbara C Paton, Klaus Harzer
    Abstract:

    Cultured skin fibroblasts from controls and patients with lysosomal storage diseases were loaded with GM1 ganglioside that had been labelled with tritium in its ceramide moiety. After a 65-h or 240-h incubation, a large percentage of this ganglioside remained undegraded in GM1 gangliosidoses, whereas in the other storage diseases studied, one of its metabolites accumulated by 2–4 fold relative to controls. Labelled GM2 ganglioside accumulated in 4 variants of GM2 gangliosidosis, whereas labelled GM3 ganglioside accumulated in sialidosis, galactosialidoses and Sphingolipid Activator Protein 1 (SAP-1, saposin B) and prosaposin (saposin A, B, C an D) deficient lipidoses. The reduced degradation of GM3 ganglioside in the SAP-1 and prosaposin deficiencies was attributed to the deficient function of SAP-1. The prosaposin deficient cells also showed a reduced re-utilization of radioactive metabolites from GM1 ganglioside (i.e. sphingosine and fatty acid) for phospholipid biosynthesis compared with fibroblasts from the SAP-1 deficient patient or normal controls. This anomaly was ascribed to the previously shown defect in ceramide degradation in prosaposin deficiency.

  • Sphingolipid Activator Protein 1 deficiency in metachromatic leucodystrophy with normal arylsulphatase a activity a clinical morphological biochemical and immunological study
    European Journal of Pediatrics, 1991
    Co-Authors: W Schlote, Barbara C Paton, Klaus Harzer, H Christomanou, B Kustermannkuhn, B Schmid, J Seeger, U Beudt, I Schuster, U Langenbeck
    Abstract:

    A 7-year-old boy had clinical features of metachromatic leucodystrophy (MLD), however, an increased urinary sulphatide excretion was found in the presence of normal arylsulphatase A (and α-galactosidase A) activity. A rectal biopsy showed metachromatically staining storage macrophages as well as nonmetachromatic, but PAS-positive, submucosal neurons filled with membranous cytoplasmic bodies. These two types of storage material led to testing for a Sphingolipid Activator Protein (SAP) deficiency. Loading tests with sulphatide and globotriaosylceramide showed deficient turnover of both Sphingolipids in cultured fibroblasts. Using the Ouchterlony method, there was no reactivity between a described anti-SAP 1 antiserum and the patient's fibroblast extracts. This new case of SAP-1 deficient MLD was compared with the four cases of this variant known from the literature. Our results indicate that rectal biopsy morphology and lipid loading biochemistry should prove useful for the screening of SAP defects.

  • immunocytochemical localization of Sphingolipid Activator Protein 2 sap 2 in normal and sap deficient fibroblasts
    European Journal of Cell Biology, 1990
    Co-Authors: Barbara C Paton, Jennifer Hughes, Klaus Harzer, A Poulos
    Abstract:

    : The intracellular localization of Sphingolipid Activator Protein 2 (SAP-2) was determined immunocytochemically using an antiserum raised against a SAP-2 preparation from Gaucher spleen. The immunolabeling indicated that SAP-2 was largely localized in the lysosomes of fibroblasts from normal individuals. In some lysosomes the labeling was greatest around the perimeter of the matrix, suggesting an association between the Activator and lysosomal membrane components. The labeling technique was also applied to fibroblasts from a patient with a unique Sphingolipid storage disorder. Consistent with immunoblotting studies on electrophoretograms, both the patient and his affected fetal sibling were found to be deficient in immunoreactive SAP-2.

B Schmid - One of the best experts on this subject based on the ideXlab platform.

  • additional biochemical findings in a patient and fetal sibling with a genetic defect in the Sphingolipid Activator Protein sap precursor prosaposin evidence for a deficiency in sap 1 and for a normal lysosomal neuraminidase
    Biochemical Journal, 1992
    Co-Authors: Barbara C Paton, A Poulos, B Kustermannkuhn, B Schmid, Klaus Harzer
    Abstract:

    It has been shown that Sphingolipid Activator Proteins (SAPs) 1 and 2 are encoded on the same gene along with two other putative Activator Proteins [Furst, Machleidt & Sandhoff (1988) Biol. Chem. Hoppe-Seyler 369, 317-328 and O'Brien, Kretz, Dewji, Wenger, Esch & Fluharty (1988) Science 241, 1098-1101]. We have undertaken further biochemical investigations on a patient and fetal sibling, who were previously shown to have a unique Sphingolipid storage disorder associated with an SAP-2 deficiency [Harzer, Paton, Poulos, Kustermann-Kuhn, Roggendorf, Grisar & Popp (1989) Eur. J. Pediatr. 149, 31-39]. The severity of their disorder suggested that other products of the SAP precursor or prosaposin gene may also be deficient. The turnover of cerebroside sulphate and globotriaosylceramide were investigated and were both impaired in fibroblasts from the patient and fetus. However, the activities of cerebroside sulphate sulphatase and globotriaosylceramide alpha-galactosidase in vitro were normal in cells from the fetus and patient respectively. In addition, there was an increase in cerebroside sulphate concentration in the kidney of the affected fetus. These results indicate that, in addition to the SAP-2 deficiency, there was a defect in SAP-1 function in this disorder. Additional increases in the concentration of monohexosyl- and dihexosyl-ceramide in the fetal kidney probably reflect the deficiency of SAP-2 in the case of monohexosylceramides, and the combined Activator deficiency in the case of dihexosylceramides. Lactosylceramide-loading studies confirmed that there was a defect in the turnover of this lipid in fibroblasts from the affected patient and fetus but not from a patient with an isolated SAP-1 deficiency, or from patients with Krabbe disease, GM1 gangliosidosis or galactosialidosis. It has been suggested [Potier, Lamontagne, Michaud & Tranchemontagne (1990) Biochem. Biophys. Res. Commun. 173, 449-456] that the prosaposin gene also codes for lysosomal neuroaminidase. However, we found normal neuraminidase activity in fibroblasts from our patient, using assay conditions which are diagnostic for sialidosis patients. The role of prosaposin gene products in Sphingolipid metabolism is discussed in view of our biochemical findings in this genetic disorder.

  • metabolism of gm1 ganglioside in cultured skin fibroblasts anomalies in gangliosidoses sialidoses and Sphingolipid Activator Protein sap saposin 1 and prosaposin deficient disorders
    Human Genetics, 1992
    Co-Authors: B Schmid, Konrad Sandhoff, Barbara C Paton, Klaus Harzer
    Abstract:

    Cultured skin fibroblasts from controls and patients with lysosomal storage diseases were loaded with GM1 ganglioside that had been labelled with tritium in its ceramide moiety. After a 65-h or 240-h incubation, a large percentage of this ganglioside remained undegraded in GM1 gangliosidoses, whereas in the other storage diseases studied, one of its metabolites accumulated by 2–4 fold relative to controls. Labelled GM2 ganglioside accumulated in 4 variants of GM2 gangliosidosis, whereas labelled GM3 ganglioside accumulated in sialidosis, galactosialidoses and Sphingolipid Activator Protein 1 (SAP-1, saposin B) and prosaposin (saposin A, B, C an D) deficient lipidoses. The reduced degradation of GM3 ganglioside in the SAP-1 and prosaposin deficiencies was attributed to the deficient function of SAP-1. The prosaposin deficient cells also showed a reduced re-utilization of radioactive metabolites from GM1 ganglioside (i.e. sphingosine and fatty acid) for phospholipid biosynthesis compared with fibroblasts from the SAP-1 deficient patient or normal controls. This anomaly was ascribed to the previously shown defect in ceramide degradation in prosaposin deficiency.

  • Metabolism of G_M1 ganglioside in cultured skin fibroblasts: anomalies in gangliosidoses, sialidoses, and Sphingolipid Activator Protein (SAP, saposin) 1 and prosaposin deficient disorders
    Human Genetics, 1992
    Co-Authors: B Schmid, B C Paton, K. Sandhoff, K. Harzer
    Abstract:

    Cultured skin fibroblasts from controls and patients with lysosomal storage diseases were loaded with G_M1 ganglioside that had been labelled with tritium in its ceramide moiety. After a 65-h or 240-h incubation, a large percentage of this ganglioside remained undegraded in G_M1 gangliosidoses, whereas in the other storage diseases studied, one of its metabolites accumulated by 2–4 fold relative to controls. Labelled G_M2 ganglioside accumulated in 4 variants of G_M2 gangliosidosis, whereas labelled G_M3 ganglioside accumulated in sialidosis, galactosialidoses and Sphingolipid Activator Protein 1 (SAP-1, saposin B) and prosaposin (saposin A, B, C an D) deficient lipidoses. The reduced degradation of G_M3 ganglioside in the SAP-1 and prosaposin deficiencies was attributed to the deficient function of SAP-1. The prosaposin deficient cells also showed a reduced re-utilization of radioactive metabolites from G_M1 ganglioside (i.e. sphingosine and fatty acid) for phospholipid biosynthesis compared with fibroblasts from the SAP-1 deficient patient or normal controls. This anomaly was ascribed to the previously shown defect in ceramide degradation in prosaposin deficiency.

  • Sphingolipid Activator Protein 1 deficiency in metachromatic leucodystrophy with normal arylsulphatase a activity a clinical morphological biochemical and immunological study
    European Journal of Pediatrics, 1991
    Co-Authors: W Schlote, Barbara C Paton, Klaus Harzer, H Christomanou, B Kustermannkuhn, B Schmid, J Seeger, U Beudt, I Schuster, U Langenbeck
    Abstract:

    A 7-year-old boy had clinical features of metachromatic leucodystrophy (MLD), however, an increased urinary sulphatide excretion was found in the presence of normal arylsulphatase A (and α-galactosidase A) activity. A rectal biopsy showed metachromatically staining storage macrophages as well as nonmetachromatic, but PAS-positive, submucosal neurons filled with membranous cytoplasmic bodies. These two types of storage material led to testing for a Sphingolipid Activator Protein (SAP) deficiency. Loading tests with sulphatide and globotriaosylceramide showed deficient turnover of both Sphingolipids in cultured fibroblasts. Using the Ouchterlony method, there was no reactivity between a described anti-SAP 1 antiserum and the patient's fibroblast extracts. This new case of SAP-1 deficient MLD was compared with the four cases of this variant known from the literature. Our results indicate that rectal biopsy morphology and lipid loading biochemistry should prove useful for the screening of SAP defects.