Prodromal substantia nigra sonography undermines suggested association between substrate accumulation and the risk for GBA-related Parkinson's disease.

BACKGROUND AND PURPOSE: Individuals with GBA (glucocerebrosidase) mutations are at increased risk of Parkinson's disease (PD). It is still debated, however, whether this increased risk results from impaired glucocerebrosidase activity leading to substrate accumulation. Comparing the presence of prodromal PD marker in GBA mutation carriers and patients with Gaucher disease (GD) (in which substrate accumulation is extensive) can assist in clarifying this issue. METHODS: In this cross-sectional study, we compared the hyperechogenic area of the substantia nigra, a prodromal PD marker, in large cohorts of GBA mutation carriers (n = 71) and patients with GD (n = 145). Our control populations were healthy, non-carriers (n = 49) and patients with GBA -related PD (n = 11). Substrate accumulation was assessed from dry blood spot levels of glucosylsphingosine. RESULTS: Our findings indicate no contribution of substrate accumulation, as the area of hyperechogenicity is similarly enlarged relative to healthy controls in both GBA mutation carriers and patients with GD. Moreover, this similarity between GBA carriers and patients with GD persists when comparing only carriers of the N370S (c.1226A>G) mutation (n = 38) with untreated patients with GD who were homozygotes for the same mutation (n = 47). In addition, measurements of hyperechogenic area did not correlate with levels of glucosylsphingosine in the untreated patients with GD. CONCLUSION: The presence of a marker of prodromal PD (substantia nigra hyperechogenicity) is independent of substrate accumulation in a population with mutated GBA . Although further longitudinal studies are needed to determine the precise predictive value of this marker for GBA -related PD, our findings raise doubts regarding the contribution of substance reduction strategies to PD prevention.

Bactericidal and cytotoxic effects of sodium hypochlorite and sodium dichloroisocyanurate solutions in vitro.

The antimicrobial and cytotoxic effects of sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) were evaluated and compared in vitro. The minimal inhibitory concentration and minimal bactericidal concentration of NaOCl and NaDCC were tested for Streptococcus sobrinus, Streptococcus salivarius, Enterococcus faecalis, and Streptococcus mutans. The cytotoxic effect was assessed by using human fibroblast tissue culture. Survival rate was assessed by a protein determination method. Results showed that the minimal inhibitory concentration and minimal bactericidal concentration values of NaOCl and NaDCC for the tested bacteria were in a similar range. NaDCC in concentrations higher than 0.02%, and NaOCl in concentrations higher than 0.01% were lethal to fibroblasts. In conclusion it seems that both agents were very effective in killing bacteria, and their cytotoxicity to fibroblasts in tissue culture was similar.

Intracellular degradation of fluorescent glycolipids by lysosomal enzymes and their activators.

Fluorescent glycolipids were utilized for detection of the intracellular, activator-dependent, activities of beta-glucocerebrosidase and arylsulphatase A. Activities were measured in primary skin fibroblasts from normal individuals, from patients with Gaucher disease who had mutations within the beta-glucocerebrosidase gene, and from a prosaposin-deficient patient. Fluorescent microscopy demonstrated that glucosylceramide or sulphatide labelled with a fluorescent probe (lissamine-rhodamine) were endocytosed and reached the lysosomes. There, in the presence of active enzyme and the corresponding saposin, they were hydrolysed to fluorescent ceramide, which changed its intracellular localization. When these substrates were labelled with pH-sensitive lissamine-rhodamine, which loses its fluorescence at neutral or alkaline pH, the transport of the product, i.e. fluorescent ceramide, from the lysosomes resulted in disappearance of the cellular fluorescence. In cells of patients having mutations within the genes encoding the glucocerebrosidase or the prosaposin, there was a considerable reduction in the intracellular rate of substrate hydrolysis that could be followed by fluorescence microscopy or measured quantitatively in cell extracts.

Ceramide toxicity and metabolism differ in wild-type and multidrug-resistant cancer cells.

Previously we demonstrated that multidrug-resistant (MDR) cancer cells have elevated levels of a glycosylated form of ceramide, glucosylceramide. Here we compared ceramide metabolism and ceramide toxicity in MCF-7 and in adriamycin-resistant (MCF-7-AdrR) human breast cancer cells. MCF-7-AdrR cells were resistant to C6-ceramide (1-10 microM); however, in MCF-7 cells treated with C6-ceramide, viability dropped sharply. Ceramide, when supplemented, was not metabolized by MCF-7 cells. In contrast, ceramide was efficiently converted to glucosylceramide by MCF-7-AdrR cells. Analysis of extracellular [3H]ceramide in radiolabeled cells showed that MCF-7-AdrR cells do not have an enhanced capacity to efflux ceramide compared with MCF-7 cells. Triphenylethylene anti-estrogens, known modulators of drug resistance, were effective inhibitors of ceramide conversion to glucosylceramide, suggesting that blocking ceramide metabolism plays a role in chemosensitization. The anti-progestine, RU486, also blocked glucosylceramide synthesis in cells; however, LY117018, a raloxifene analog, was without influence. We propose that an enhanced capacity to glycosylate ceramide as evidenced in MCF-7-AdrR cells, is a molecular determinant of drug resistance, particularly as regards resistance to ceramide-enhancing agents such as anthracyclines, ionizing radiation, and tumor necrosis factor-alpha.

Importance of splicing for prosaposin sorting.

The prosaposin gene encodes a 70 kDa protein. This protein might either reach the lysosomes and get processed there to four peptides, which are activators of known lysosomal enzymes, or be secreted by cells as a 70 kDa protein, recently anticipated to have several biological activities. The human prosaposin gene has a 9 bp exon (exon 8) that is alternatively spliced, thus encoding three prosaposin forms: one with an extra three amino acid residues, one with an extra two residues and a third form with no extra residues. With the aim of testing whether there is an association between the alternative splicing and the differential sorting of prosaposins, we cloned two human prosaposin cDNA forms in a T7/EMC/vaccinia virus-derived vector and expressed them in human cells. The results indicated that the prosaposin containing the three extra residues accumulated faster and in greater amounts in the medium, whereas the prosaposin with no extra residues was mainly destined for lysosomes. Point mutations created by mutagenesis in vitro in the 9 bp stretch had a diverse effect on prosaposin secretion. When supplied to cells in the medium, both prosaposins were endocytosed and reached the lysosomes, where they were processed to active saposin B and saposin C. The activities of the saposins were monitored qualitatively and quantitatively. Quantitatively, lipids were extracted from the cells, separated on TLC and measured fluorimetrically. Qualitatively, cells were detected by fluorescence microscopy.

Intracellular degradation of sulforhodamine-GM1: use for a fluorescence-based characterization of GM2-gangliosidosis variants in fibroblasts and white blood cells.

A novel fluorescent ganglioside, sulforhodamine-GM1 was administered into cells derived from carriers and patients with different subtypes of GM2 gangliosidosis, resulting from various mutations in the gene encoding the lysosomal enzyme hexosaminidase (Hex) A. The cells used were skin fibroblasts and white blood cells, i.e. lymphocytes, monocytes and macrophages. In the severe infantile form of the GM2 gangliosidosis, Tay-Sachs disease, the sulforhodamine-GM1 was hydrolyzed within the lysosomes to the corresponding sulforhodamine-GM2 which, because of lack of Hex A activity, was not further degraded. In comparison, in the cells derived from GM2 gangliosidoses carriers, as well as pseudodeficient and adult forms of GM2 gangliosidosis, the sulforhodamine-GM2 was further processed and sequentially degraded by the lysosomal glycosidases to sulforhodamine-ceramide. The latter was converted to sulforhodamine-sphingomyelin, which was secreted into the culture medium. The fluorescence of the sulforhodamine ceramide in cell extracts and/or sulforhodamine-sphingomyelin in the culture medium was quantified and related to parallel data obtained using cells of normal individuals. This permitted distinguishing between the various GM2 gangliosidoses subtypes and relating the intracellular hydrolysis of sulforhodamine-GM1 to the genotypes of the respective GM2 gangliosidoses variants.

Fluorescence-based selection of retrovirally transduced cells in the absence of a marker gene: direct selection of transduced type B Niemann-Pick disease cells and evidence for bystander correction.

Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from the deficient activity of acid sphingomyelinase (ASM). Type A NPD is characterized by the absence of residual ASM activity, massive accumulation of sphingomyelin and cholesterol within lysosomes, and a rapid, neurodegenerative course that leads to death by 3 years of age. In contrast, type B NPD patients have low, but detectable, levels of residual ASM activity and little or no neurologic disease. Thus, individuals with type B NPD may survive into late adolescence or adulthood and are considered excellent candidates for somatic cell gene therapy. To facilitate the development of gene therapy for this disorder, a novel procedure was devised to isolate metabolically corrected type B NPD cells in the absence of marker gene expression. Type B NPD cells were transduced with retroviral vectors expressing ASM, labeled with lissamine rhodamine sphingomyelin (LR-SPM), and subjected to preparative fluorescence-activated cell sorting (FACS). Two non-overlapping cell populations were isolated, corresponding to enzymatically corrected (i.e., low fluorescence) and noncorrected (i.e., high fluorescence) cells. Quantitative PCR analysis demonstrated that the enzymatically corrected cells were enriched for vector sequences. Moreover, the corrected cells could be regrown and continued to express high levels of ASM activity after numerous passages, consistent with the fact that they were stably transduced. Notably, coculture of FACS-sorted, overexpressing cells with untreated type B NPD fibroblasts resulted in a homogeneous cell population with low fluorescence whose FACS distribution overlapped that of the corrected cells. Computerized fluorescence microscopy confirmed that nearly all of these cocultured cells expressed ASM activity and could hydrolyze LR-SPM.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnosing sphingolipidoses in murine and human embryos.

The aim of this study was to diagnose lipid storage diseases in embryos at the preimplantation stage. Two parallel approaches were employed. Firstly, activities of several sphingolipid hydrolases were determined in extracts of murine embryos and also human oocytes and polyspermic embryos. Sensitive fluorescent or fluorogenic procedures provided indications that Tay-Sachs, Gaucher and Krabbe diseases might be diagnosed in one human blastomere, while for Niemann-Pick disease two might be required. Secondly, pyrene lipids were administered into murine embryos and their fluorescence was quantified by computerized imaging microscopy. As a model of Gaucher disease, the fluorescent substrate pyrene glucosylceramide was administered into murine embryos in the presence or absence of an inhibitor of the enzyme beta-glucosidase. Because of decreased degradation of the substrate in enzyme-inhibited cells, the fluorescence per blastomere was considerably greater relative to those which received no inhibitor. The results indicated that lipid storage diseases might be diagnosed in single human blastomeres at the preimplantation stage, obviating the need for pre-natal diagnosis and abortion of affected foetuses.

Toward gene therapy for Niemann-Pick disease (NPD): separation of retrovirally corrected and noncorrected NPD fibroblasts using a novel fluorescent sphingomyelin.

The neurologic (type A) and nonneurologic (type B) forms of Niemann-Pick disease (NPD) both result from deficiencies of acid sphingomyelinase (ASM) activity leading to the accumulation of sphingomyelin and other related lipids within lysosomes. Recently, the full-length cDNA and genomic sequences encoding ASM have been isolated and the nature of the molecular lesions causing NPD has been investigated. Although these developments have facilitated diagnosis for this debilitating disease, no effective treatment is currently available. Toward this latter goal, our laboratories recently reported the effectiveness of retroviral-mediated gene transfer for the in vitro correction of the cellular pathology in NPD fibroblasts (Suchi et al., 1992). In addition, novel selection procedures were developed to separate retrovirally corrected and noncorrected NPD fibroblasts based on the receptor-mediated delivery of a fluorescently (pyrene)-labeled sphingomyelin (P12-SPM) to the lysosomes of cells using liposomes coated with apolipoprotein E. In this study, we have used a different, fluorescent derivative of sphingomyelin (lissamine-rhodamine dodecanoyl sphingomyelin; LR12-SPM) to extend and improve this selection system. LR12-SPM offers a number of advantages over P12-SPM, including the facts that apolipoprotein E is not required for its efficient uptake and targeting to lysosomes and that the product of LR12-SPM degradation by ASM is efficiently transported out of cells. Thus, when analyzed in a fluorescence-activated cell sorter (FACS), there was complete separation (i.e., no overlap) of retrovirally corrected and noncorrected NPD cells after the administration of LR12-SPM.(ABSTRACT TRUNCATED AT 250 WORDS)

Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.

Types A and B Niemann-Pick disease (NPD) result from inherited deficiencies of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12). To evaluate the feasibility of somatic gene therapy for the treatment of these disorders, retroviral-mediated gene transfer was used to introduce the full-length ASM cDNA into cultured fibroblasts from two unrelated type A NPD patients. The ASM activities in these cells were less than 4% of mean normal levels, and, consequently, they accumulated approximately 3-fold elevated levels of sphingomyelin. After retroviral-mediated transfer of the ASM cDNA, ASM activities in the NPD cells increased to levels up to 16-fold those found in normal fibroblasts. In addition, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies also were undertaken to evaluate the effects of retroviral-mediated gene transfer on the pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a type A NPD patient by using apolipoprotein E-mediated endocytosis, only approximately 6% of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced (i.e., "corrected") by retroviral-mediated gene transfer could degrade approximately 80% of the delivered sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the pathology of NPD cells. Cell-loading studies were also used to develop a selection system for discriminating between NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene-labeled sphingomyelin, was 3- to 5-fold that of normal or transduced cells. As a consequence, the NPD and transduced cells could be efficiently sorted by flow cytometry with a fluorescence-activated cell sorter. In addition, the NPD cells could be selectively killed by photosensitization after irradiation with a long-wavelength UV light. These results should permit direct selection of ASM-expressing cells after retroviral-mediated gene transfer without the need to preselect for a cotransferred marker gene.

Administration of pyrene lipids by receptor-mediated endocytosis and their degradation in skin fibroblasts.

Sphingomyelin and seven glycosphingolipids were labeled with the fluorescent probe pyrene and administered into cultured fibroblasts by receptor-mediated endocytosis. For this purpose pyrene sphingomyelin or mixtures of pyrene glycolipid and unlabeled sphingomyelin were dispersed as small, unilamellar liposomes. Apolipoprotein E was then added and the receptor for this ligand on the cell surface was utilized for uptake of the liposomes and their transport to the lysosomes, where the respective pyrene lipids were degraded. Following incubation with each of the respective pyrene lipids, only the administered compound and the pyrene ceramide were present; intermediate hydrolysis products were not detected. This indicated that, in skin fibroblasts, the lysosomal ceramidase was limiting and controlled the rate of total degradation of the pyrene sphingolipids.

Photosensitization of cultured cells and viruses by pyrene lipids.

Administration of pyrene-linked fatty acids and lipids to cultured cells or an enveloped (vesicular stomatitis) virus induced photosensitization which, following irradiation with a long ultra-violet light (LUV), resulted in killing of the cells and loss of the infectivity of the virus with the following specific effects. (i) LUV illumination of the pyrene-sphingomyelin administered cultured skin fibroblasts derived from normal individuals and patients with Niemann-Pick disease permitted selective killing of the latter. (ii) Similarly LUV illumination of pyrenedodecanoic acid (P12) incubates of leukemic cell lines mixed with human bone marrow cells permitted selective killing of the former. (iii) LUV illumination of P12 incubates of vesicular stomatitis virus decreased the infectivity of the virus by up to 12 logs.

Human acid beta-glucosidase: inhibition studies using glucose analogues and pH variation to characterize the normal and Gaucher disease glycon binding sites.

Comparative kinetic studies with glycon inhibitors were used to investigate the properties of the active site of human acid beta-glucosidase (EC 3.2.1.45) from normal placenta and spleens of type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the pure normal enzyme, the specificity of glycon binding was assessed with 35 glucose derivatives and epimers. Most glycons were mixed type inhibitors with a predominantly competitive nature (i.e., Kis much less than Kii) and had low apparent affinity for the enzyme (Kisapp = 20-500 mmol/l). beta-Glucose-1-phosphate was unusual, since it inhibited 4-methylumbelliferyl-beta-glucoside hydrolysis in an uncompetitive pattern (Kiapp = 0.55 mmol/l) but had no effect on glucosyl ceramide hydrolysis. C-1- (1-deoxy-1-amino-beta-D-glucose) and C-3- (3-deoxy-3-amino-D-glucose) amino and C-5-imino [1-deoxynojirimycin (dNM), nojirimycin and castanospermine] substituted sugars were highly potent inhibitors with Kisapp(beta-glucose)/Kisapp approximately equal to 10(3)-10(5); an amine at C-2 did not alter Kisapp compared to beta-glucose. The variation of Kisapp with pH for the 5-imino- and 1-deoxy-1-aminoglycosides conformed to a model for the unprotonated inhibitors binding to the protonated forms (EH and EH2) of the diprotic (Vmaxapp and Vmaxapp/Kmapp) normal enzyme (pK1 = 4.7; pK2 = 6.7) with pH-independent Kisapp values of 2.9-9.0 mumol/l and 0.22 mmol/l, respectively. Several of the amine-containing inhibitors competitively protected the enzyme from inactivation by conduritol B epoxide, a covalent active site-directed inhibitor, indicating interaction with residues at that site. With the partially purified AJGD splenic enzymes, the results were the same except that Kisapp(AJGD)/Kisapp(normal) = 4-17 for dNM and 1-deoxy-1-amino-beta-glucose; this ratio was approximately equal to 1 with most other glycons, and particularly, nojirimycin and castanospermine. The results of these studies indicated that the glycon binding site of the normal acid beta-glucosidase contains important residues for interaction with the C-2, C-3 and C-4 hydroxyl groups of beta-glucose and a residue with pKa = 6.7 which was critical to the binding of amine-containing inhibitors and the hydrolysis of substrates. The findings were consistent with a specific alteration in or near the glycon binding site which results in the functional abnormalities of the mutant AJGD acid beta-glucosidase.

Human acid beta-glucosidase. Use of conduritol B epoxide derivatives to investigate the catalytically active normal and Gaucher disease enzymes.

Human acid beta-glucosidase (glucosylceramidase; EC 3.2.1.45) cleaves the glycosidic bonds of glucosyl ceramide and synthetic beta-glucosides. Conduritol B epoxide (CBE) and its brominated derivative are mechanism-based inhibitors which bind covalently to the catalytic site of acid beta-glucosidase. Procedures using brominetritiated CBE and monospecific anti-human placental acid beta-glucosidase IgG were developed to determine the molar concentrations of functional acid beta-glucosidase catalytic sites in pure placental enzyme preparations from normal sources; kcat values then were calculated from Vmax = [Et]kcat using glucosyl ceramide substrates with dodecanoyl (2135 +/- 45 min-1) and hexanoyl (3200 +/- 410 min-1) fatty acid acyl chains and 4-alkyl-umbelliferyl beta-glucoside substrates with methyl (2235 +/- 197 min-1), heptyl (1972 +/- 152 min-1), nonyl (2220 +/- 247 min-1), and undecyl (773 +/- 44 min-1) alkyl chains. The respective kcat values for acid beta-glucosidase in a crude normal splenic preparation were about 60% of these values. In comparison, the kcat values of the mutant splenic acid beta-glucosidase from two Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were about 1.5-3-fold decreased and had Km values for each substrate which were similar to those for the normal acid beta-glucosidase. The interaction of the normal and Type 1 AJGD enzymes with CBE in a 1:1 stoichiometry conformed to a model with reversible EI complexes formed prior to covalent inactivation. With CBE, the equal kmax values (maximal rate of inactivation) for the normal (0.051 +/- 0.009 min-1) and Type 1 AJGD (0.058 +/- 0.016 min-1) enzymes were consistent with the minor differences in kcat. In contrast, the Ki value (dissociation constant) (839 +/- 64 microM) for the Type 1 AJGD enzymes was about 5 times the normal Ki value (166 +/- 57 microM). These results indicated that the catalytically active Type 1 AJGD acid beta-glucosidase had nearly normal hydrolytic capacity and suggested an amino acid substitution in or near the acid beta-glucosidase active site leading to an in vivo instability of the mutant enzymatic activity.

Human acid beta-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site.

Human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic beta-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3H-labeled conduritol B epoxide (3H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V8 protease digestion of the 3H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (Mr, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid beta-glucosidase.

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.

Genetic heterogeneity in Gaucher disease: physicokinetic and immunologic studies of the residual enzyme in cultured fibroblasts from non-neuronopathic and neuronopathic patients.

To elucidate the genetic heterogeneity in the three major phenotypic subtypes of Gaucher disease, the residual acid beta-glucosidase in fibroblasts from patients with all three subtypes from different ethnic and demographic groups was investigated by comparative kinetic, thermostability, and immunotitration studies. The kinetic studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to the enzyme modifiers, taurocholate (or phosphatidylserine), and glucosyl sphingosine (or N-hexyl glucosyl sphingosine); Group A residual enzymes responded normally to these modifiers. All neuronopathic patients (types 2 and 3) and most non-Jewish, non-neuronopathic patients (type 1) had group A residual activities and thus could not be distinguished by their kinetic properties. Group B residual enzymes had markedly abnormal responses to these modifiers. All Ashkenazi and only two non-Jewish type 1 patients had group B residual activities. Group C residual activity had an intermediate response to all modifiers and represented a single Afrikaner type 1 patient. Pedigree studies indicated that this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. Thermostability studies showed additional heterogeneity of the residual activities within the three kinetic groups. Group A (type 2) and group B (type 1) enzymes had similarly decreased thermostabilities. In contrast, group A (type 1) residual activities were heterogeneous; three classes of thermostabilities were found among these enzymes: normal, decreased, and increased. Immunotitration of equal amounts of the normal or Gaucher disease beta-glucosidase activities with monospecific IgG indicated that the enzyme proteins from most Gaucher disease patients were antigenically altered and/or that large amounts of catalytically abnormal or inactive antigen were present. A decreased amount of antigenically and catalytically normal enzyme was present in a group A, type 1 African black patient, suggesting decreased stability or synthesis of his mutant acid beta-glucosidase. These kinetic, immunologic, and thermostability studies indicated that 1) type 1 Gaucher disease is biochemically heterogeneous and results from at least four distinct allelic acid beta-glucosidase mutations that alter enzyme structure and/or function, 2) neuronopathic and non-Jewish non-neuronopathic phenotypes cannot be distinguished reliably by kinetic analyses alone, and 3) the Ashkenazi type 1 Gaucher disease results from a unique mutation that alters a specific active site domain of acid beta-glucosidase.

Gaucher disease types 1, 2, and 3: differential mutations of the acid beta-glucosidase active site identified with conduritol B epoxide derivatives and sphingosine.

To elucidate the genetic heterogeneity in Gaucher disease, the residual beta-glucosidase in cultured fibroblasts from affected patients with each of the major phenotypes was investigated in vitro and/or in viable cells by inhibitor studies using the covalent catalytic site inhibitors, conduritol B epoxide or its bromo derivative, and the reversible cationic inhibitor, sphingosine. These studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to these inhibitors. Group A residual enzymes had normal I50 values (i.e., the concentration of inhibitor that results in 50% inhibition) for the inhibitors and normal or nearly normal t1/2 values for conduritol B epoxide. All neuronopathic (types 2 and 3) and most non-Jewish nonneuronopathic (type 1) patients had group A residual activities and, thus, could not be distinguished by these inhibitor studies. Group B residual enzymes had about four- to fivefold increased I50 values for the inhibitors and similarly increased t1/2 values for conduritol B epoxide. All Ashkenazi Jewish type 1 and only two non-Jewish type 1 patients had group B residual activities. The differences in I50 values between groups A and B also were confirmed by determining the uninhibited enzyme activity after culturing the cells in the presence of bromo-conduritol B epoxide. Group C residual activity had intermediate I50 values for the inhibitors and represented a single Afrikaner type 1 patient: this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. These inhibition studies indicated that: Gaucher disease type 1 is biochemically heterogeneous, neuronopathic and non-Jewish nonneuronopathic phenotypes cannot be reliably distinguished by these inhibitor studies, and the Ashkenazi Jewish form of Gaucher disease type 1 results from a unique mutation in a specific active site domain of acid beta-glucosidase that leads to a defective enzyme with a decreased Vmax.