Immunological and catalytic quantitation of splenic glucocerebrosidase from the three clinical forms of Gaucher disease.

The enzymatic activity of glucocerebrosidase in splenic extracts of the adult nonneurological form of Gaucher disease (type I) was 15% +/- 7% of normal, and the titer of enzyme cross-reacting material (ECRM) in these spleens was 54% +/- 9% of normal. The titer of ECRM in splenic extracts of tissues obtained from patients with the neurological forms of Gaucher disease (types II and III) was essentially the same as in type I Gaucher spleens (59% +/- 10% of normal), but the measurable catalytic activity of glucocerebrosidase in these spleens was substantially lower than that found in type I Gaucher spleens (2.3% +/- 0.6% of normal). Thus, the attentuated glucocerebrosidase activity in spleens from all three forms of Gaucher disease appears to stem from a structurally mutated enzyme that is altered in its catalytic efficiency and possibly in its antigenic expression.     

Clinical phenotype of Gaucher disease in relation to properties of mutant glucocerebrosidase in cultured fibroblasts.

We have investigated several parameters of glucocerebrosidase in cultured skin fibroblasts from patients with various clinical phenotypes of Gaucher disease. In this study no strict correlation was found between the clinical manifestations of Gaucher disease and the parameters investigated in fibroblasts. These parameters included the specific activity of the enzyme in extracts towards natural lipid and artificial substrate in the presence of different activators; the enzymic activity per unit of glucocerebrosidase protein; the rate of synthesis of the enzyme and its stability; and the post-translational processing of the enzyme. In addition, the activity in situ of glucocerebrosidase in fibroblasts was investigated using a novel method by analysis of the catabolism of NBD-glucosylceramide in cells that were loaded with bovine serum albumin-lipid complexes. Again, no complete correlation with the clinical phenotype of patients was detectable. Glucocerebrosidase in fibroblasts from most non-neuronopathic (type 1) Gaucher disease patients differs in some aspects from enzyme in cells from patients with neurological forms (types 2 and 3). The stimulation by activator protein and phospholipid is clearly more pronounced in type 1 than in types 2 and 3; the enzymic activity per unit of glucocerebrosidase protein in type 1 is severely reduced in the presence of taurocholate and the amount of glucocerebrosidase appears (near) normal in contrast to the situation in types 2 and 3 Gaucher fibroblasts. However, this distinction was not always consistent; glucocerebrosidase in fibroblasts from some type 1 Gaucher patients, particularly some South African cases, was comparable in properties to enzyme in type 2 and 3 patients.     

Comparative study on glucocerebrosidase in spleens from patients with Gaucher disease.

In Gaucher disease (glucosylceramide lipidosis), deficiency of glucocerebrosidase causes pathological storage of glucosylceramide, particularly in the spleen. A comparative biochemical and immunological analysis has therefore been made of glucocerebrosidase in spleens from normal subjects (n = 4) and from Gaucher disease patients with non-neuronopathic (n = 5) and neuronopathic (n = 5) phenotypes. The spleens from all Gaucher disease patients showed markedly decreased glucocerebrosidase activity. Discrimination of different phenotypes of Gaucher disease was not possible on the basis of the level of residual enzyme activity, or by measurements, using the immunopurified enzyme, of kinetic constants, pI or molecular mass forms. A severe decrease was found in the specific activity of glucocerebrosidase purified to homogeneity from the spleen of a patient with the non-neuronopathic phenotype of Gaucher disease, as compared with that of the enzyme purified from the spleen of a normal subject. This finding was confirmed by an immunological method developed for accurate assessment of the relative enzyme activity per molecule of glucocerebrosidase protein. The method revealed that the residual enzyme in the spleens of all investigated patients with a non-neuronopathic course of Gaucher disease had a more than 7-fold decreased activity of glucocerebrosidase (measured in the presence of taurocholate) per molecule of enzyme, and that the concentration of glucocerebrosidase molecules in the spleens of these patients was near normal. Observations made with immunoblotting experiments were consistent with these findings. In contrast, in the spleens of patients with neuronopathic phenotypes of Gaucher disease, the concentration of glucocerebrosidase molecules was severely decreased.     

Gaucher disease: The effects of phosphatidylserine on glucocerebrosidase from normal and Gaucher fibroblasts

Summary Glucocerebroside -glucosidase (glucocerebrosidase) activity was assayed from cultured fibroblasts of normal individuals, and patients with type 1 (non-neuropathic), type 2 (acute neuropathic), and type 3 (subacute neuropathic) form of Gaucher disease. Residual glucocerebrosidase activity of patients was 8.9 to 17.4% of normal controls, and there was no clear correlation between the level of residual enzyme activity and the different clinical subtypes of the disease. When membrane-bound glucocerebrosidase activity was assayed in the presence of crude brain lipid extracts or purified phosphatidylserine, enzyme from both the normal and type 1 Gaucher fibroblasts was stimulated dramatically (35–60% by crude extracts, 85–90% by phosphatidylserine). This stimulation was not observed with fibroblast glucocerebrosidase of an infantile type 2 and two juvenile type 3 Gaucher patients. The presence of inhibitors of glucocerebrosidase in these type 2 and type 3 Gaucher cells was not detected. Contrary to the mutant enzyme from these Gaucher fibroblasts, glucocerebrosidase from fibroblasts of two adult type 3 Gaucher patients with cerebral involvement was stimulated substantially (72–85%) by phosphatidylserine. When membrane-bound glucocerebrosidase from fibroblasts of the infantile type 2 and juvenile type 3 patients was solubilized with sodium cholate (1% w/v) and delipidated, the phospholipid stimulation of enzyme activity was restored. These findings suggest that considerable clinical and biochemical heterogeneity exists among patients with neuropathic Gaucher disease and that phosphatidylserine activation cannot be used as a reliable indicator in predicting future onset of neurodegeneration in Gaucher patients. The possibility of an aberrant binding of mutant glucocerebrosidase to the lysosomal membrane in juvenile type 3 form of Gaucher disease is discussed.     

Substrate reduction therapy: clinical evaluation in type 1 Gaucher disease

Glycosphingolipid (GSL) lysosomal storage disorders are inherited enzyme deficiencies that result in pathological lysosomal accumulation of glycolipids, with widespread clinical consequences. Type 1 Gaucher disease is the commonest of these; the deficient enzyme in this condition is glucocerebrosidase. Clinical manifestations include hepatosplenomegaly, thrombocytopenia, anaemia, recurrent infections and skeletal lesions. The condition can be treated with intravenous enzyme replacement therapy (ERT). Substrate reduction therapy is a new approach in which glycolipid accumulation is counteracted not by replacing the deficient enzyme but by reducing the substrate level to better balance residual activity of the deficient enzyme. Miglustat is an inhibitor of glucosylceramide synthase, a key enzyme in GSL synthesis. Oral administration of miglustat to patients with type 1 Gaucher disease attenuates the synthesis of glucocerebroside, the substrate of the deficient glucocerebrosidase. In the first clinical study, patients with type 1 Gaucher disease who had enlargement of the liver or spleen and (if present) the spleen at baseline received 12 months treatment with oral miglustat. There were mean decreases in liver and spleen volumes of 12% (7.9-16.4, p < 0.001) and 19% (14.3-23.7, p < 0.001), respectively. Mean haemoglobin increased by 0.26 g dl(-1) (-0.5-0.57, not statistically significant) and platelet count by 8.3 x 10(9) l(-1) (1.9-14.7, p = 0.014).     

Delivery,Distribution, and Neuronal Uptake of Exogenous Mannose-Terminal Glucocerebrosidase in the Intact Rat Brain

Gaucher disease is caused by insufficient activity of the enzyme glucocerebrosidase. Great benefit has been obtained through enzyme replacement therapy for patients with type 1 (non-neuronopathic) Gaucher disease. In contrast, inconsistent effects of enzyme therapy have been observed in patients with type 3 (chronic neuronopathic) Gaucher disease, and no benefit on the lethal course of the disease occurs in patients with Type 2 (acute neuronopathic) Gaucher disease. We examined the use of convection-enhanced delivery to augment the delivery and distribution of exogenous glucocerebrosidase (m.w. 63,000) to the brain by infusing it under slight hydrostatic pressure into the striatal region of rats. The enzyme was comparatively stable under these conditions. It was distributed from the site of injection toward the cerebral cortex where it became primarily localized in neurons. These findings provide considerable incentive for the exploration of intracerebral microinfusion of enzyme to the brain of patients with metabolic storage disorders involving the CNS.     

Properties of acid ceramidase from human spleen

We have characterised ceramidase activity in extracts of human spleen from control subjects and from patients with Gaucher disease. In Triton X-100 extracts of control spleens, a broad pH optimum of pH 3.5-5.0 was found; no ceramidase activity was detectable at neutral or alkaline pH. About 45-60% of acid ceramidase could be extracted from spleen without detergents, but for complete extraction, Triton X-100 was required. For the radiolabelled substrate oleoylsphingosine, a Km of 0.22 +/- 0.09 mM and a Vmax of 57 +/- 11 nmol/h per mg protein was calculated in spleen from a control subject. Flat-bed isoelectric focussing in the presence of Triton X-100 revealed a pI of 6.0-7.0 for acid ceramidase; similar values were found for sphingomyelinase and glucerebrosidase. HPLC-gel filtration indicated that in the presence of Triton X-100, acid ceramidase has an Mr of about 100 kDa. In the absence of detergents, the enzyme forms high-molecular-weight aggregates. Similar aggregation behaviour was observed for sphingomyelinase, while the elution of beta-hexosaminidase was not affected by detergents. The elution profile of glucocerebrosidase was only slightly altered by Triton X-100. There was no difference in the properties of acid ceramidase present in spleen from control subjects and from patients with type I Gaucher disease.     

Hydrophilic iminosugar active-site-specific chaperones increase residual glucocerebrosidase activity in fibroblasts from Gaucher patients

Gaucher disease is an autosomal recessive lysosomal storage disorder caused by the deficient activity of glucocerebrosidase. Accumulation of glucosylceramide, primarily in the lysosomes of cells of the reticuloendothelial system, leads to hepatosplenomegaly, anemia and skeletal lesions in type I disease, and neurologic manifestations in types II and III disease. We report herein the identification of hydrophilic active-site-specific chaperones that are capable of increasing glucocerebrosidase activity in the cultured fibroblasts of Gaucher patients. Screening of a variety of natural and synthetic alkaloid compounds showed isofagomine, N-dodecyl deoxynojirimycin, calystegines A3, B1, B2 and C1, and 1,5-dideoxy-1,5-iminoxylitol to be potent inhibitors of glucocerebrosidase. Among them, isofagomine was the most potent inhibitor of glucocerebrosidase in vitro, and the most effective active-site-specific chaperone capable of increasing residual glucocerebrosidase activity in fibroblasts established from Gaucher patients with the most prevalent Gaucher disease-causing mutation (N370S). Intracellular enzyme activity increased approximately two-fold after cells had been incubated with isofagomine, and the increase in glucocerebrosidase activity was both dose-dependent and time-dependent. Western blotting demonstrated that there was a substantial increase in glucocerebrosidase protein in cells after isofagomine treatment. Immunocytochemistry revealed an improvement in the glucocerebrosidase trafficking pattern, which overlaps that of lysosome-associated membrane protein 2 in Gaucher fibroblasts cultivated with isofagomine, suggesting that the transport of mutant glucocerebrosidase is at least partially improved in the presence of isofagomine. The hydrophilic active-site-specific chaperones are less toxic to cultured cells. These results indicate that these hydrophilic small molecules are suitable candidates for further drug development for the treatment of Gaucher disease.     

Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease

Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type of the disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases.     

Electrophoresis of glucocerebrosidase from normal and Gaucher disease fibroblasts.

A polyacrylamide gel electrophoresis system for glucocerebrosidase has been developed. This method was used to characterize the glucocerebrosidase activity of normal and Gaucher disease fibroblasts; the residual glucocerebrosidase activity in adult Gaucher disease fibroblasts co-migrates with the activity from normal fibroblasts.     

A kinetic study of the effects of galactocerebroside 3-sulphate on human spleen glucocerebrosidase. Evidence for two activator-binding sites.

Extraction of control human spleen glucocerebrosidase with sodium cholate and butan-l-ol reversibly inactivates the enzyme in terms of its ability to hydrolyse the water-soluble substrate 4-methylumbelliferyl beta-D-glucopyranoside (MUGlc). The acidic brain lipid galactocerebroside 3-sulphate (sulphatide) reconstitutes beta-glucosidase activity in a strongly concentration-dependent manner. In this study we show that sulphatide exhibits three critical micellar concentrations (CMCs): CMC1, 3.72 microM; CMC2, 22.6 microM; CMC3, 60.7 microM. We designate the aggregates formed at these CMCs as primary, secondary and tertiary micelles respectively. From the results of kinetic studies performed at various sulphatide concentrations (0.012-248 microM), we found that sulphatide monomers (less than 3 microM) decreased the Km (for MUGlc) of control glucocerebrosidase from 11 to 4.6 mM, and lowered the Vmax. 2-fold. However, secondary and tertiary micelles were required for expression of high control glucocerebrosidase activities. Glucocerebrosidase prepared from the spleen of a patient with non-neuronopathic type 1 Gaucher's disease exhibited a very low Km (2.8 mM) even in the absence of exogenous lipid, and sulphatide monomers had no effect on the mutant enzyme's Km or Vmax. However, secondary or tertiary micelles markedly increased the Vmax. of the type 1 glucocerebrosidase to 60% of the corresponding control enzyme value. In contrast, for the glucocerebrosidase of the neuronopathic type 2 case, although sulphatide decreased the Km from 9.2 to 1.7 mM, the Vmax. never reached more than 5% that of the control enzyme, even at high concentrations of sulphatide. In addition, we found that secondary and tertiary sulphatide micelles enhanced the rate of inactivation of all three glucocerebrosidase preparations by chymotrypsin. Collectively, these results indicate the presence of two sulphatide-binding sites on glucocerebrosidase: one that enhances substrate binding, and another that enhances catalysis.     

Uptake of Mannose-Terminal Glucocerebrosidase in Cultured Human Cholinergic and Dopaminergic Neuron Cell Lines

Enzyme replacement therapy has been shown to be particularly effective for patients with type 1 (non-neuronopathic) Gaucher disease. However, intravenously administered glucocerebrosidase does not reverse or halt the progression of brain damage in patients with type 2 (acute neuronopathic) Gaucher disease. A previous investigation revealed that intracerebral infusion of mannose-terminal glucocerebrosidase was safe in experimental animals. The enzyme had a comparatively long half-life in the brain. It was transported by convection from the site of infusion along white matter fiber tracts to the cerebral cortex where it was endocytosed by neurons. In anticipation of intracerebral administration of mannose-terminal glucocerebrosidase to patients with type 2 Gaucher disease, it was important to learn the mechanism involved in its cellular uptake. We therefore compared the endocytosis of this enzyme by J774 macrophage cells with that in two human neuronal cell lines and a human astrocyte cell line. Mannose-terminal glucocerebrosidase was taken up by cholinergic LA-N-2 cells, but to a much lower extent than by macrophages. Considerably less of the enzyme was endocytosed by dopaminergic SH-SY5Y cells. It was not taken up by NHA astrocytes. The findings provide encouragement for an exploration of intracerebral administration of glucocerebrosidase in patients with type 2 Gaucher disease.     

Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its implications for Parkinson disease

Gaucher disease, caused by pathological mutations GBA1, encodes the lysosome-resident enzyme glucocerebrosidase, which cleaves glucosylceramide into glucose and ceramide. In Gaucher disease, glucocerebrosidase deficiency leads to lysosomal accumulation of substrate, primarily in cells of the reticulo-endothelial system. Gaucher disease has broad clinical heterogeneity, and mutations in GBA1 are a risk factor for the development of different synucleinopathies. Insights into the cell biology and biochemistry of glucocerebrosidase have led to new therapeutic approaches for Gaucher disease including small chemical chaperones. Such chaperones facilitate proper enzyme folding and translocation to lysosomes, thereby preventing premature breakdown of the enzyme in the proteasome. This review discusses recent progress in developing chemical chaperones as a therapy for Gaucher disease, with implications for the treatment of synucleinopathies. It focuses on the development of non-inhibitory glucocerebrosidase chaperones and their therapeutic advantages over inhibitory chaperones, as well as the challenges involved in identifying and validating chemical chaperones.     

Characterization of glucocerebrosidase in peripheral blood cells and cultured blastoid cells

We have characterized glucocerebrosidase in various cell types of peripheral blood of control subjects and in cultured human blastoid cells. The intracellular level of glucocerebrosidase in cultured blastoid cells (10-30 nmol substrate hydrolyzed/h.mg protein) resembles closely values observed for leukocyte cell types and various tissues and is significantly lower than that observed in cultured fibroblasts (150-500 nmol substrate hydrolyzed/h.mg protein). Glucocerebrosidase is extracted from leukocyte cell types and cultured blastoid cells almost exclusively in a monomeric, nonactivated form with enzymatic properties identical to those of the tissue enzyme. In contrast, extracts of platelets are rich in an aggregated, activated form of the enzyme. Glucocerebrosidase in blood cells and cultured blastoid cells is heterogeneous with respect to Mr and pI due to a heterogeneous oligosaccharide composition of the enzyme. The different forms seen represent intermediates in the biosynthesis and maturation of the enzyme. Blastoid cells should thus be an attractive model system for studying the natural history of glucocerebrosidase in a cell type related to those cells involved in the pathology of Gaucher disease.     

Gene therapy of Gaucher's and Fabry's diseases: current status and prospects

Gaucher disease and Fabry disease are lysosomal storage disorders characterized by the accumulation of sphingolipids. In both cases, the goal of gene therapy is to permanently provide tissues with enzyme levels allowing to avoid storage of the undigested substrates. Different gene therapy strategies must however be designed as Gaucher disease is due to a deficiency in the membrane-associated enzyme glucocerebrosidase, whereas Fabry disease is caused by a deficiency in the soluble enzyme alpha-galactosidase. Indeed, a soluble enzyme can be provided to tissues is trans by gene-modified cells whereas gene transfer has to target the most affected cells in the case of membrane-bound enzymes. Thus, in non-neurological Gaucher disease (type 1), the hematopoietic tissue has to be targeted as the deficiency affects the monocyte/macrophage lineage. Following promising preclinical studies, clinical protocols have been initiated to explore the feasibility and safety of retroviral transfer of the glucocerebrosidase gene into CD34+ cells from patients with type 1 Gaucher disease. Although gene-marked cells were detected in vivo, the level of corrected cells was very low, a finding indicating that improved vectors along with partial myeloablation may be necessary. Here, lentiviral vectors should enable more gene transduction into the hematopoietic target cells. As concerns the diffuse neurological lesions in types 2 and 3 of Gaucher disease, they will probably be especially difficult to target by gene therapy because of the non soluble nature of glucocerebrosidase. Finally, over the last few years, Fabry disease has become a compelling target for gene therapy as an etiology-based treatment strategy. Indeed, several recent studies aiming at creating a large in vivo source of alpha-galactosidase have yielded positive long-term results in the Fabry knock-out mouse model.     

Failure of alglucerase infused into Gaucher disease patients to localize in marrow macrophages.

BACKGROUND: Gaucher disease is a common glycolipid storage disease, caused by a deficiency of lysosomal beta-glucosidase (glucocerebrosidase). Alglucerase is a form of glucocerebrosidase enriched with terminal mannose moieties, so as to "target" the preparation to the high-affinity macrophage receptor in patients with Gaucher disease. Our earlier in vitro studies indicated that alglucerase was bound by cells other than macrophages by a widely distributed, low-affinity mannose receptor. MATERIALS AND METHODS: Bone was removed at surgery from six patients with Gaucher disease; in three cases, bone was obtainable both when the patient was untreated and after receiving an infusion of alglucerase. Four samples of bone were obtained from patients without Gaucher disease and served as controls. A bone marrow aspirate was obtained from another patient with Gaucher disease immediately after enzyme infusion. Marrow beta-glucosidase activity and chitotriosidase (a macrophage marker) was determined on all samples. RESULTS: Even with the large bolus doses used for the treatment of Gaucher disease by some, scarcely any beta-glucosidase activity was found in marrow samples; the amount of the enzyme was much less than would have been anticipated had the enzyme been evenly distributed to all body cells. CONCLUSIONS: Alglucerase is not targeted to marrow macrophages. Its unquestioned therapeutic effectiveness must be due either to its activity at some site other than marrow macrophages or to the fact that the doses administered are so enormous that even a small fraction is sufficient to achieve a therapeutic effect.     

A new glucocerebrosidase-gene missense mutation responsible for neuronopathic Gaucher disease in Japanese patients.

We have identified a new T-to-A single-base substitution at nucleotide 3548 (in the genomic sequence) in exon 6 in the glucocerebrosidase gene from a patient with Gaucher disease type 3. This mutation caused a substitution of isoleucine for phenylalanine at amino acid residue 213 (of 497 residues in the mature protein). By in vitro expression study in cultured mammalian cells, this mutation resulted in deficient activity of glucocerebrosidase. By allele-specific oligonucleotide hybridization of selectively PCR-amplified DNA from eight unrelated Japanese Gaucher disease patients, this mutant allele was observed in other neuronopathic Japanese Gaucher disease patients, in moderately frequent occurrence (three of six neuronopathic patients). This observation suggests that this allele was one of severe [corrected] alleles which were related to the development of neurological manifestations of Gaucher disease.     

Purification and properties of a heat-stable glucocerebrosidase activating factor from control and Gaucher spleen.

Gaucher's disease is a lysosomal storage disease caused by a deficiency in the enzyme glucocerebrosidase. A small, heat-stable glycoprotein first obtained from Gaucher spleen (Ho, M. W., and O'Brien, J. S. (1971) Proc. Natl. Acad. Sci. U. S.A. 68, 2810-2813) has been observed to stimulate the activity of glucocerebrosidase isolated from normal tissue. It has been suggested that this material might be important in the physiological catabolism of glucocerebroside in normal individuals (Ho, M. W. (1974) in Enzyme Therapy in Lysosomal Storage Diseases (Tager, J. M., Hooghwinkel, G. J. M., and Daems, W. Th., eds) pp.239-246, North-Holland Publishing Co., Amsterdam). In order to investigate this suggestion, glucocerebrosidase activating factors were isolated and purified from control and Gaucher spleen and characterized. Although approximately the same mass of activator was isolated from both spleens, the two activators differ from one another in a number of important respects: (a) the activator from the control spleen is only 6 per cent as active (on a protein basis) as the activator from Gaucher spleen; (b) the amino acid compositions of the purified activators are significantly different; and (c) carbohydrate analysis of the purified activators indicates that the activator from Gaucher spleen is a glycoprotein, while that from control spleen is not. Comparative kinetic studies demonstrate that the anionic detergent, sodium taurocholate, and the acidic phospholipid, phosphatidylinositol, both stimulate glucocerebrosidase activity to a larger extent than the activator substance from Gaucher spleen. The activator from Gaucher spleen and human liver glucocerebrosidase both appear to contain significant hydrophobic character. We conclude that the activator is probably not physiologically important in the catabolism of glucocerebroside in normal tissues. The significance of the occurrence of this apparently unique glycoprotein activator in Gaucher spleen remains obscure; however, its presence represents another interesting aspect of Gaucher's disease that warrants further investigation.     

Biosynthesis and maturation of glucocerebrosidase in Gaucher fibroblasts

The biosynthesis and maturation of glucocerebrosidase were studied in fibroblasts from patients with the neurological and non-neurological forms of Gaucher disease and in control cells. In control fibroblasts the precursor of glucocerebrosidase (62-63 kDa), observed after a short pulse with [35S]methionine, was converted during the chase period to a 66-kDa intermediate form and, finally, to the 59-kDa mature protein. In fibroblasts from patients with the non-neurological phenotype of Gaucher disease (type 1) the same biosynthetic forms were seen as in control fibroblasts. These biosynthetic forms correspond to the three-banded pattern seen in control and Gaucher type 1 fibroblast extracts analysed by the immunoblotting procedure, or after electrophoresis and fluorography of extracts of such fibroblasts cultured for 5 days with [14C]leucine. The 59-kDa protein seen in type 1 fibroblasts was unstable and disappeared after a prolonged chase; this disappearance was not observed when the cells were grown in the presence of leupeptin. In fibroblasts from patients with the neurological forms of Gaucher disease (types 2 and 3) the 62.5-kDa precursor of glucocerebrosidase was present in near-normal amounts after a short pulse, but the 59-kDa form was not detected even when cells were cultured with leupeptin. These results are in accordance with the absence of the 59-kDa band in immunoblots of types 2 and 3 fibroblast extracts. Culturing of type 1, type 2 and type 3 Gaucher fibroblasts in the presence of leupeptin led to an increase in the activity of glucocerebrosidase.     

Activation of human spleen glucocerebrosidases by monoacylglycol sulfates and diacylglycerol sulfates

The study of the acidic lipid requirement of human spleen glucocerebrosidase was extended to include two new series of acidic lipids, namely, monoacylglycol sulfates and diacylglycerol sulfates. Lysosomal glucocerebrosidase was extracted with sodium cholate and 1-butanol to render its beta-glucosidase activity dependent upon exogenous lipids. Maximum reactivation of control glucocerebrosidase was obtained with nonanoylglycol sulfate (NGS) and diheptanoylglycerol sulfate (DHGS). However, the effects of these lipids were markedly dependent on the nature of buffer used in the assay medium; specifically, 0.2 M sodium citrate-phosphate (pH 5.5) was much more effective than 0.2 M sodium acetate (pH 5.5) in permitting these lipids to reactivate glucocerebrosidase. In contrast, the marked activation of glucocerebrosidase by phosphatidylserine and galactocerebroside 3-sulfate (sulfatide) that was achievable in the sodium acetate buffer was totally inhibited by citrate or phosphate ions. The effects of NGS and DHGS on the kinetic parameters of control glucocerebrosidase were to lower the Km for the substrate, 4-methylumbelliferyl-beta-D-glucoside from 5.5 mM to approximately 2 mM (in sodium citrate-phosphate buffer) and markedly increase the Vmax. Furthermore, with DHGS, significant activation was achieved at concentrations below the lipid's critical micellar concentration. None of the monoacylglycol- or diacylglycerol sulfates were capable of stimulating mutant glucocerebrosidases from either type 1 (Ashkenazi-Jewish) or type 2 Gaucher's disease patients. Like control glucocerebrosidase, the type 1 glucocerebrosidase was unresponsive to phosphatidylserine and sulfatide when the beta-glucosidase assay was conducted in 0.2 M sodium citrate-phosphate buffer. Based on the differential action of these lipid activators in the two buffers and their effects on the mutant enzymes, we propose that, with regard to the lipid requirement of glucocerebrosidase, there are two classes of acidic lipids--one comprised of phosphatidylserine and sulfatide and the other comprised of the likes of NGS, DHGS, or sodium taurodeoxycholate. It appears that control glucocerebrosidase and the mutant enzyme of the patient with type 1 Gaucher's disease is reconstitutable with the first class of lipids whereas the glucocerebrosidase of the type 2 patient is not. The observations in this report are interpreted in terms of a model which postulates that normal glucocerebrosidase possesses at least two distinct lipid binding domains.