g(m1)-ganglioside and Gaucher-Disease

Topics: Adolescent; Adult; Animals; beta-Galactosidase; Cats; Cerebrovascular Disorders; Child, Preschool; Coronary Disease; Fabry Disease; Female; G(M1) Ganglioside; G(M2) Ganglioside; Gangliosidoses; Gaucher Disease; Glycolipids; Hexosaminidases; Humans; Infant; Infant, Newborn; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Male; Sodium-Potassium-Exchanging ATPase; Vascular Resistance

The lipid composition or the liver, spleen, brain, cerebellum and cerebrospinal fluid of a Gaucher disease type II patient who died at the age of 5 months was examined. The glycolipid analysis demonstrated a marked increase of total amounts not only in the peripheral tissues but also in the brain cerebellum and cerebrospinal fluid, with a prevalence of glucosylceramide. A reduction in gangliosides was observed in all the analysed tissues with a relative increase of GD3 in the nervous tissue. The fatty acid composition of glucosylceramide showed a prevalence of stearic acid in the central nervous system, while in the peripheral tissues palmitic acid was prevalent. This result suggests a different origin of the glucosylceramide stored in different tissues. The generalized reduction of gangliosides and their modified distribution together with the central nervous system GD3 increment represent a new observation. These data could be useful in the effort to clarify the pathophysiological mechanism of brain damage in neuronopathic Gaucher disease.

Topics: Brain; Brain Chemistry; Cerebellum; Female; G(M1) Ganglioside; Gangliosides; Gaucher Disease; Glucosylceramides; Glycolipids; Humans; Infant; Lactosylceramides; Liver; Spleen

Saposin B is involved in the hydrolysis of sulfatides, GM1 ganglioside, globotriaosylceramide, and several other sphingolipids and glycerolipids by lysosomal hydrolases. Saposin B is one of four small glycoproteins (saposins) derived from prosaposin. The carbohydrate chain of saposin B was removed and deglycosylated saposin B was characterized and compared with native saposin B. Deglycosylated saposin B stimulated the enzymatic hydrolysis of ganglioside GM1 by acid beta-galactosidase and sulfatide by arylsulfatase A to the same extent as native saposin B. In addition deglycosylated saposin B bound sulfatide and GM1 ganglioside identical to native saposin B. The stability of native saposin B to proteolytic digestion was unchanged by deglycosylation. Neither native saposin B nor deglycosylated saposin B were hydrolyzed by trypsin, endoproteinase Glu-C (V-8), chymotrypsin, or a mixture of acid proteases isolated from human testis. Unlike its effect on metabolic stability, the carbohydrate chain appears to affect folding of saposin B. When native and deglycosylated saposin B were reduced under denaturing conditions and refolded under identical conditions examination of the refolded products indicated that each protein was refolded in a qualitatively different way. A human mutation in saposin B-deficient metachromatic leukodystrophy, in which its glycosylation site is eliminated, has been reported. Our observations suggest that instability of the mutated saposin B is not due to the absence of a protective effect of the carbohydrate chain on proteolysis, but is likely due to aberrant folding resulting from the absence of a carbohydrate chain.

Topics: beta-Galactosidase; Carbohydrates; Cerebroside-Sulfatase; Chromatography, High Pressure Liquid; Drug Stability; Electrophoresis, Polyacrylamide Gel; G(M1) Ganglioside; Gaucher Disease; Glycoproteins; Glycosylation; Humans; Hydrolysis; Saposins; Sphingolipid Activator Proteins; Spleen; Structure-Activity Relationship; Sulfoglycosphingolipids

We show that sialosylgangliotetraosylceramide (GM1) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:beta-glucosidase. Stimulation of 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis by the beta-glucosidase was markedly dependent upon the concentration of GM1 in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of GM1 above 18 degrees C; the CMC of the primary micelles (3.32 microM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 microM at 37 and 20 degrees C, respectively). In the temperature range of 18-39 degrees C, beta-glucosidase activity increased sharply when the GM1 concentration was above the CMC of the secondary micelles. Although a heat-stable factor, purified from the spleen of a patient with Gaucher's disease, had a profound effect on the activation of beta-glucosidase by GM1, it decreased the CMC only slightly (14.8 versus 17.2 microM at 37 degrees C). The heat-stable factor (8 micrograms/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits beta-glucosidase to be activated by primary micelles or monomers. The results of gel filtration chromatography and sucrose gradient centrifugation in H2O and D2O revealed that the activation of beta-glucosidase by GM1 was associated with an increase in the size of the enzyme from 45,800 to 178,500 daltons and an increase in the partial specific volume from 0.697 to 0.740 ml/g. The active, reconstituted beta-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of GM1 below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37 degrees C, indicating that GM1 monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside by the enzyme.

Topics: Animals; beta-Glucosidase; Chemical Phenomena; Chemistry, Physical; Dose-Response Relationship, Drug; Enzyme Activation; G(M1) Ganglioside; Gangliosides; Gaucher Disease; Glucosidases; Glucosides; Hot Temperature; Humans; Hymecromone; Inositol; Kinetics; Liver; Lysosomes; Male; Micelles; Molecular Weight; Rats; Rats, Inbred Strains; Temperature

An improved method, which combined a number of published techniques, is described for the polyethylene-glycol-induced fusion of mononuclear human skin fibroblasts in the presence of phytohaemagglutinin-P and for the subsequent isolation of polynuclear cells by Ficoll gradient sedimentation. Enriched cultures contain between 60 and 75% multinucleated cells and may be maintained in culture without fetal calf serum for up to 14 days without significant overgrowth by the few contaminating mononuclear parental cells. Complementation appears not to occur between GM1 gangliosidosis and mucopolysaccharidosis, type VI B (Morquio) cell strains; this experimental observation provides support for the earlier hypothesis that the mutations for these conditions are allelic. Earlier observations that complementation does not occur between selected phenotypic variants (viz., neuronopathic forms and those without neurological involvement) of sphingomyelin storage (Niemann-Pick) disease or Gaucher's disease are confirmed.

Topics: Cell Fusion; Cells, Cultured; Enzymes; Fibroblasts; G(M1) Ganglioside; Gangliosidoses; Gaucher Disease; Genetic Complementation Test; Humans; Lysosomes; Mucopolysaccharidosis IV; Niemann-Pick Diseases

Glucocerebrosidase from normal human spleen, and spleen from cases of neurologic (types 2 and 3) and nonneurologic (type 1) Gaucher's disease, was delipidated and inactivated by extraction from membranes with sodium cholate and ice-cold 1-butanol. Control glucocerebrosidase was stimulated markedly by large quantities (20-30 micrograms/assay) of phosphatidylserine (PS), or by a combination of smaller amounts (1-2 micrograms) of PS and 3 micrograms of a heat-stable factor (HSF) derived from the spleen of a patient with Gaucher's disease. The residual glucocerebrosidase from a nonneurologic case, but not a neurologic case, was also responsive to PS and HSF. The combination of HSF and PS decreased the Km of the normal enzyme for 4-methylumbelliferyl-beta-D-glucopyranoside from 8.0 to 1.6 mM. These effectors also increased the reactivity of glucocerebrosidase to the inhibitor conduritol B epoxide; HSF alone had no effect (t1/2 = 19 +/- 0.5 min) whereas the maximum rate of inactivation (t1/2 = 4.0 min) by conduritol B epoxide was achieved in the presence of a mixture of PS (1 microgram) and HSF (3 micrograms). Phosphatidylglycerol (PG) and phosphatidic acid, also acidic phospholipids, were effective activators of glucocerebrosidase. Varying the fatty acid composition of PG had little effect on its ability to stimulate glucocerebrosidase activity. However, in the case of phosphatidylcholine (PC), a weaker activator than PG or PS, fatty acid composition had a significant impact on the ability of this neutral lipid to activate glucocerebrosidase; dilinoleoyl-PC and dicaproyl-PC were moderately effective activators, but distearoyl-PC and dioleoyl-PC were almost totally inactive. The mono-, and di-, and trisialogangliosides (GM1, GD1, and GT1 were less than half as effective as PS as activators of glucocerebrosidase. These results indicate that acidic phospholipids and the heat-stable factor may both play a role in explaining the genetic heterogeneity of Gaucher's disease.

Topics: Enzyme Activation; Fatty Acids; G(M1) Ganglioside; Gaucher Disease; Glucosidases; Glucosylceramidase; Humans; Kinetics; Lipids; Phosphatidylserines; Reference Values; Spleen; Structure-Activity Relationship

Topics: Adult; beta-Glucosidase; Cells, Cultured; Enzyme Activation; Fibroblasts; G(M1) Ganglioside; G(M2) Ganglioside; Gangliosides; Gaucher Disease; Glucosidases; Hot Temperature; Humans; Isoelectric Focusing; Male; Skin

Topics: beta-Galactosidase; beta-Glucosidase; Cell Membrane; Chromatography, Gel; G(M1) Ganglioside; Gangliosidoses; Gaucher Disease; Glucosidases; Glucosylceramidase; Hot Temperature; Humans; Isoelectric Focusing; Spleen; Substrate Specificity

Peculiar storage cells appearing in bone marrow aspirates from a patient with juvenile GM1-gangliosidosis and from one with beta-thalassemia were examined light microscopically, histochemically and electron microscopically. Light microscopically, most of the storage cells closely resembled Gaucher cells pathognomonic for Gaucher's disease. The cytoplasm of the Gaucher-like cells contained numerous variable-shaped membrane-bound inclusions mostly arranged in a mosaic pattern and filled with fibrillar materials. Intermingled tubular structures were usually narrow as compared to those of the Gaucher cells. These ultrastructural differences of the stored materials between the Gaucher-like cells and Gaucher cells were more clearly substantiated by the high resolution electron microscopy with negative staining technique. Enzyme cytochemically, acid phosphatase activity was proved in or around the storage inclusions, suggesting their lysosomal origin. Histochemically, it might be suggested that the stored materials of the Gaucher-like cells in juvenile GMI-gangliosidosis were non-sulfated acid mucopolysaccharides and glycopeptides, whereas glycoproteins were the major component of the storage cells in beta-thalassemia. Possible mechanisms of storage in the Gaucher-like cells were discussed in both disorders.

Topics: Child; Erythrocytes; Female; G(M1) Ganglioside; Gangliosides; Gangliosidoses; Gaucher Disease; Glycosaminoglycans; Histocytochemistry; Humans; In Vitro Techniques; Inclusion Bodies; Male; Periodic Acid-Schiff Reaction; Thalassemia

Topics: Acid Phosphatase; Adult; Amino Acids; Child; Child, Preschool; Enzyme Activation; Female; G(M1) Ganglioside; Galactosidases; Gaucher Disease; Glucosidases; Glucosylceramidase; Glycoproteins; Hot Temperature; Humans; Infant; Male; Molecular Weight; Spleen