Molecular basis of GM1 gangliosidosis and Morquio disease, type B. Structure-function studies of lysosomal beta-galactosidase and the non-lysosomal beta-galactosidase-like protein.

GM1 gangliosidosis and Morquio B disease are distinct disorders both clinically and biochemically yet they arise from the same beta-galactosidase enzyme deficiency. On the other hand, galactosialidosis and sialidosis share common clinical and biochemical features, yet they arise from two separate enzyme deficiencies, namely, protective protein/cathepsin A and neuraminidase, respectively. However distinct, in practice these disorders overlap both clinically and biochemically so that easy discrimination between them is sometimes difficult. The principle reason for this may be found in the fact that these three enzymes form a unique complex in lysosomes that is required for their stability and posttranslational processing. In this review, I focus mainly on the primary and secondary beta-galactosidase deficiency states and offer some hypotheses to account for differences between GM1 gangliosidosis and Morquio B disease.

Purification of G-M-1-ganglioside and ceramide lactoside beta-galactosidase from rabbit brain.

The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.

Classification of disorders of GM2 ganglioside hydrolysis using 3H-GM2 as substrate.

Rates of GM2 ganglioside hydrolysis by fibroblasts from normal controls and patients with GM2 gangliosidosis were measured in situ, with cells growing in tissue culture by assaying the decrease in cell-incorporated 3H-GM2 over time, and in vitro by assaying the rate of 3H-GM2 hydrolysis using fibroblast extracts in the presence of no additives, sodium taurocholate, and GM2 activator protein. In tissue culture, normal cells hydrolyzed cell-incorporated GM2 while fibroblasts from patients with GM2 gangliosidosis did not. The half life of GM2 in normal fibroblasts was 78 hours. In vitro, only normal fibroblast extracts hydrolyzed GM2 in the absence of additives. In the presence of 10 mM sodium taurocholate, rates of GM2 hydrolysis by normal fibroblast extracts were increased 5-16-fold, fibroblast extracts from AB and B1 variant patients hydrolyzed GM2 at normal rates, cell extracts from patients with Tay-Sachs disease hydrolyzed GM2 at nearly normal rates, and cell extracts from Sandhoff disease patients hydrolyzed GM2 at about 10% of normal rates. In the presence of 1 microgram of GM2 activator, rates of GM2 hydrolysis by normal fibroblast extracts were increased 8-25-fold, fibroblast extracts from a patient with the AB variant hydrolyzed GM2 at normal rates, and cell extracts from other variants of GM2 gangliosidosis did not hydrolyze GM2. The results suggest that measuring the persistence of 3H-GM2 in tissue culture over time will detect any variant of GM2 gangliosidosis and may be the ideal way to test for the presence of this disease. Variants can be distinguished by assaying the hydrolysis of 3H-GM2 using cell extracts in the absence of additives, with sodium taurocholate, and with activator.

The biochemistry and clinical features of galactosialidosis.

Galactosialidosis is a heterogeneous disorder that is manifested in infantile, late infantile, juvenile/adult, and atypical forms. In every instance the primary defect is in the ability of protective protein to associate with beta-galactosidase and neuraminidase to protect them from intralysosomal proteolysis. The protective protein is in reality a serine protease that displays both cathepsin A and C-terminal deamidase activity. We summarize the major clinical features of each form, and the range of storage products accumulated. The concept of an intralysosomal complex containing beta-galactosidase and neuraminidase in addition to protective protein seems irrefutable but major gaps exist in our understanding of how the complex is formed and in what subcellular organelles, how it is sustained, and the protein domains contributed by the constituent enzymes that play a pivotal role in this process.

Enzymatic hydrolysis of sphingomyelin liposomes by normal tissues and tissues from patients with Niemann-Pick disease.

Liposomes of [3H]sphingomyelin are readily hydrolyzed by extracts of human spleen, liver, cultured skin fibroblasts and purified placental sphingomyelinase in the absence of detergents. The pH optimum for hydrolysis by liver and spleen extracts was 6.5-7.0 while the fibroblast activity showed an optimum at pH 4.0-4.3. However, the pH optimum for purified placental sphingomyelinase in the presence of Triton X-100 (pH 5.0) is only slightly different from that displayed with liposomes (pH 5.3). The data clearly show that hydrolysis of liposomal sphingomyelin by sphingomyelinase is affected by the composition and purity of the enzyme source.

Studies on the activation of the enzymatic hydrolysis of sphingomyelin liposomes.

Two pH optima were observed for the hydrolysis of sphingomyelin liposomes by brain and fibroblast extracts; one at pH 4.2-4.5, the other at pH 7-8. The proportion of the acidic activity in fibroblasts was affected greatly by the culturing conditions. Both the acidic and neutral enzyme activities were deficient in Niemann-Pick Type A fibroblasts, suggesting that both were genetically related. Partially purified activators from normal as well as Gaucher disease spleen stimulated the hydrolysis of sphingomyelin, at both pH values, by fibroblast and brain extracts. After further purification by DE-52 and Sephacryl 200 column chromatography the Gaucher activator retained its ability to stimulate sphingomyelinase and was active as well towards beta-glucocerebrosidase and beta-galactocerebrosidase.

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100.

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U

Concanavalin A promotes the uptake of lysosomal hydrolases by human fibroblasts.

Human placental hexosaminidase B and beta-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4 degrees C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37 degrees C is refractory to haptens treatment. These results are interpreted as an initial binding of enzyme to concanvalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.

Aggregation of plasma Z type alpha 1-antitrypsin suggests basic defect for the deficiency.

The abnormal type of alpha 1-antitrypsin, PI (protease inhibitor) type Z, is associated with inclusion bodies in the liver, which contain non-secreted alpha 1-antitrypsin. Our studies show that Z protein has an inherent tendency to aggregate, even in plasma. Depending upon conditions, from 15 to 70% of the Z protein in plasma was in a high-Mr form, compared with 1.5% of M type alpha 1-antitrypsin. The high-Mr complex in plasma cannot be disaggregated using Triton X detergent or reducing conditions. This increased tendency to aggregate can be explained by the mutation affecting, tertiary structure and salt bridge formation in Z protein. We have observed this same tendency to aggregate for Mmalton alpha 1-antitrypsin, a rarer variant also associated with a plasma deficiency.

Maturation and degradation of beta-galactosidase in the post-Golgi compartment are regulated by cathepsin B and a non-cysteine protease.

Lysosomal beta-galactosidase precursor is processed to a mature form and associated with protective protein in lysosomes. In this study we used two cysteine protease proinhibitors, E64-d for cathepsins B, S, H, and L, and CA074Me for cathepsin B. They are converted intracellularly to active forms, E-64c and CA074, respectively. Both active compounds inhibited maturation of the exogenous beta-galactosidase precursor, but E-64c did not inhibit further degradation to an inactive 50-kDa product. We concluded that cathepsin B participated exclusively in maturation of beta-galactosidase, and a non-cysteine protease was involved in further degradation and inactivation of the enzyme molecule.

Deficiency of phospholipase C acting on phosphatidylglycerol in Niemann-Pick disease.

We examined the degradation of a labeled phosphatidylglycerol (PG) by fibroblasts from a normal control and a patient with Niemann-Pick (NP) disease. The control homogenate had both phospholipase A and phospholipase C activities toward PG, but NP cells had only phospholipase A. The PG phospholipase C of control fibroblasts was solubilized by sonication and freezing and thawing, was most active at pH 5.0, and was inhibited by Ca2-, detergents, sphingomyelin, and 5' AMP. Assay of PG phospholipase C in fibroblast cultures from NP patients with sphingomyelinase deficiency (three designated type A and four type B) confirmed absence of activity, whereas cultures from NP patients without sphingomyelinase deficiency (three designated type C and one with neurovisceral lipidoses and vertical supranuclear ophthalmoplegia) had activities close to those of normal controls. These findings substantiate previous observations of low phosphodiesterase activities in NP disease and suggest that the enzymatic function affected by the NP genes includes specificity toward PG and sphingomyelin. Deficiency of PG phospholipase C may explain the accumulation of bis(monoacylglycero)phosphate in NP disease.

Type 2 GM1 gangliosidosis with long survival and neuronal ceroid lipofuscinosis.

Neurologic deterioration began in a girl before age 2 years. By 4 she was spastic and decerebrate. GM1 gangliosidosis was diagnosed by absence of beta-galactosidase activity in leukocytes and fibroblasts. She died at 17 years. Her small brain contained only 2.61 mumole glycolipid N-acetylneuraminic acid per gram, and was filled with autofluorescent material. GM1 gangliosidosis was confirmed by the presence of membranous cytoplasmic bodies, by the absence of beta-galactosidase, and by failure of complementation when the patient's fibroblasts were fused with cells from other forms of GM1 gangliosidosis. The autofluorescent material probably accumulated because of the long survival rather than the primary enzyme defect.

Early-infantile galactosialidosis: prenatal presentation and postnatal follow-up.

Galactosialidosis (GS) is an autosomal recessive condition caused by combined deficiency of the lysosomal enzymes beta-galactosidase and alpha-neuraminidase. The combined deficiency has been found to result from a defect in protective protein/cathepsin A (PPCA), an intralysosomal protein which protects these enzymes from premature proteolytic processing. The most severe form of GS, the early-infantile form, results in early onset of edema, ascites, visceromegaly, and skeletal dysplasia. We report a case of early-infantile GS in a male infant who presented with nonimmune fetal hydrops (NIH), "coarse" facial appearance, massive fluid-filled inguinal hernias, multiple telangiectasia, and diffuse hypopigmentation; he subsequently developed visceromegaly. The diagnosis of GS was confirmed biochemically and the defect in PPCA characterized at the protein level. Examination of fetal peripheral blood smears sampled at 30 weeks gestation demonstrated vacuolation of lymphocytes, suggesting blood film examination may be a useful screening tool for cases of NIH where a metabolic disorder is suspected. Skeletal radiography at birth demonstrated punctate epiphyses of the femora, calcanei, and sacrum. We present a discussion of and differential diagnosis for this radiographic finding. To the best of our knowledge, this is the first case of early-infantile GS presenting with stippled epiphyses.

Late-onset Tay-Sachs disease presenting as catatonic schizophrenia: diagnostic and treatment issues.

BACKGROUND: It is not commonly appreciated that patients with hexosaminidase A deficiency (Tay-Sachs disease) can first present in adulthood with psychiatric illness. METHOD: A 17-year-old non-Jewish male patient was referred with a history of treatment-resistant catatonic schizophrenia. We uncovered coexistent neurologic abnormalities and evidence of cognitive decline. Metabolic screening revealed a severe deficiency of beta-hexosaminidase A. We reviewed the literature on late-onset gangliosidosis with attention to (1) the nature of the associated psychiatric and neurologic abnormalities and (2) the treatment of psychosis in these patients. RESULTS: The patient's catatonia responded promptly to benzodiazepine therapy. Treatment with neuroleptic medication resulted in the rapid development of neuroleptic malignant syndrome. The patient was thereafter maintained on lorazepam with resolution of his acute psychiatric disturbances and unexpected improvement in his neurologic status. CONCLUSION: Patients with hexosaminidase deficiency may first present in adolescence or adulthood with psychiatric illness, particularly schizophrenic-like psychosis. The presence of speech disturbance, gait abnormalities, movement disorders, and cognitive decline may indicate an underlying metabolic disorder. The use of traditional neuroleptics to treat the psychosis in such individuals may produce an unacceptably high risk/benefit ratio. Benzodiazepines may ameliorate the psychiatric and neurologic abnormalities in these patients.

First trimester prenatal diagnosis of Tay-Sachs disease using the sulfated synthetic substrate for hexosaminidase A.

Uncultured and cultured embryonic trophoblastic tissue obtained by chorionic villus sampling (CVS) displays enzyme activity towards 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranosyl-6-sulfate (MUGS), a specific substrate for Hexosaminidase A (Hex A), the enzyme deficient in Tay-Sachs disease (TSD). Specific activity is comparable to that found in cultured amniocytes and fibroblasts. The enzyme activity has a pH optimum of 4.1 and an apparent Km of 6 x 10(-4) mol/L. Thirteen pregnancies in eight families at risk for TSD were monitored by CVS using MUGS as the Hex A substrate. Four fetuses were proven affected by enzyme analysis of fetal tissues and cultured fetal fibroblasts obtained at the time of termination of the pregnancies. Nine fetuses were judged to be unaffected. Eight babies were clinically normal while the other pregnancy is continuing. The use of MUGS as substrate for Hex A makes prenatal diagnosis by CVS of families at risk for TSD simple, direct and accurate.

Selective damage to cochlear efferents by the choline neurotoxin ethylcholine mustard aziridinium ion (AF64A) in the chinchilla.

The cholinotoxin ethylcholine mustard aziridinium ion (AF64A) was diluted in artificial perilymph to concentrations ranging from 10-100 microM, injected unilaterally into the bulla of chinchillas, and allowed to passively diffuse across the round window membrane. Following 21-day survival, the animals were sacrificed and ears removed and embedded in epoxy for histological evaluation under both light and transmission electron microscopy. At 10 microM concentration, selective degeneration of efferent fibers was observed in the efferent terminals on outer hair cells (OHC), tunnel radial fibers, tunnel spiral bundle, and the inner spiral bundle. Serial sections of the middle turn of an animal at 10 microM concentrations showed normal efferent terminals on approximately 50% of OHCs. At the higher concentrations non-specific damage was seen in OHCs, afferents, and some supporting cells. These data suggest that low doses AF64A produces selective damage to cochlear efferent terminals and fibers in the chinchilla.

Monoclonal antibodies against human placental sphingomyelinase.

Monoclonal antibodies against human placental acid sphingomyelinase have been raised. The antibodies are all of the IgG1 type, and are quite specific for the enzyme. One of the antibodies has been used to demonstrate a common antigenic identity of three polypeptides (mol.wts. 90,000, 72,000, and 48,000) of a purified sphingomyelinase preparation.

Caprine beta-D-mannosidosis: characterization of a model lysosomal storage disorder.

Interest in using caprine beta-D-mannosidosis as a model to evaluate bone marrow transplantation in the treatment of human lysosomal storage disorders provided the stimulus for characterization of beta-D-mannosidase in selected goat tissues and induction of hemopoietic chimerism in the goat. Total beta-D-mannosidase activity was measured with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. Residual activity in mutant liver was 52% of control but no activity was detectable in mutant kidney or brain tissue. Normal adult goat liver contained two forms of beta-D-mannosidase, a nonlysosomal form (52%) with a broad pH range for optimum activity (4.5-8.0) and a lysosomal form (48%) with a pH optimum of 5.5. Residual enzyme in mutant liver consisted entirely of the nonlysosomal form. Normal adult thyroid, kidney and brain contained two major lysosomal isoenzymes with pIs 5.5 and 5.9 and traces of a minor isoenzyme with pI 5.0. Normal liver contained three isoenzymes with similar pIs; however, an isoenzyme with pI 5.0 predominated. In 60-day fetal liver lysosomal isoenzymes predominated and only trace amounts of nonlysosomal isoenzyme were detectable. Total hepatic beta-D-mannosidase activity increased towards adult levels during the last 90 days of gestation as a result of increasing nonlysosomal isoenzyme activity. Intraperitoneal injection of fetal liver cells into 60-day goat fetuses resulted in sustained hemopoietic chimerism in surviving kids without evidence of graft-versus-host-disease. These results suggest that transplantation of normal fetal liver cells into preimmunocompetent goat fetuses affected with beta-D-mannosidosis is feasible and may provide an alternative strategy for evaluation of postnatal bone marrow transplantation in the treatment of human lysosomal storage disorders.