Acid Beta-glucosidase Activity Research Articles

Antiviral activity of acid beta-glucosidase 1 on enterovirus 71, a causative agent of hand, foot and mouth disease.

Enterovirus 71 (EV71) is a causative agent of hand, foot and mouth disease (HFMD). EV71 causes fever, rash, diarrhoea and, in some cases, acute encephalopathy/encephalitis, which can be fatal. No specific treatment is currently available for EV71 infection. Here, we conducted a cDNA library screen and identified acid β-glucosidase 1 (GBA1; also known as β-glucocerebrosidase) as an EV71 resistance factor. The anti-EV71 function of GBA1 was verified by gene transduction and knockdown experiments. Cerezyme, a molecular drug used to treat Gaucher's disease and having recombinant human GBA1 as the active ingredient, protected against EV71 infection. The anti-EV71 activity of GBA1 was bimodal: endogenous GBA1 restricted cell surface expression levels of scavenger receptor class B, member 2 (SCARB2), also known as lysosomal integral membrane protein 2 (LIMP-2), and exogenous recombinant GBA1 interfered with EV71 to interact with SCARB2 outside the cell. Thus, our findings suggest that GBA1 may represent a novel molecular target for the treatment of EV71 infection.

Profile of eliglustat tartrate in the management of Gaucher disease.

Gaucher disease (GD) is a lysosomal storage disorder caused by the deficient activity of acid beta glucosidase, with consequent accumulation of glucosylceramide in the spleen, liver, bone marrow, and various organs and tissues. Currently, the gold standard for GD treatment is enzyme replacement therapy (ERT). The efficacy of ERT in improving or stabilizing the visceral and hematological symptoms of GD is well-proven. However, since ERT has to be administered by frequent intravenous infusions, this therapeutic approach has an important impact on the patient’s quality of life. Eliglustat tartrate is a new substrate reduction therapy for GD, which acts as a specific and potent inhibitor of glucosylceramide synthase and can be administered orally. This review summarizes the results of the preclinical and clinical trials, which experimented with eliglustat, and discusses its possible role in the management of GD, when compared to the currently available treatments and the new experimental approaches.

Combined saposin C and D deficiencies in mice lead to a neuronopathic phenotype, glucosylceramide and α-hydroxy ceramide accumulation, and altered prosaposin trafficking

Saposins (A, B, C and D) are approximately 80 amino acid stimulators of glycosphingolipid (GSL) hydrolases that derive from a single precursor, prosaposin. In both humans and mice, prosaposin/saposin deficiencies lead to severe neurological deficits. The CD-/- mice with saposin C and D combined deficiencies were produced by introducing genomic point mutations into a critical cysteine in each of these saposins. These mice develop a severe neurological phenotype with ataxia, kyphotic posturing and hind limb paralysis. Relative to prosaposin null mice ( approximately 30 days), CD-/- mice had an extended life span ( approximately 56 days). Loss of Purkinje cells was evident after 6 weeks, and storage bodies were present in neurons of the spinal cord, brain and dorsal root ganglion. Electron microscopy showed well-myelinated fibers and axonal inclusions in the brain and sciatic nerve. Marked accumulations of glucosylceramides and alpha-hydroxy ceramides were present in brain and kidney. Minor storage of lactosylceramide (LacCer) was observed when compared with tissues from the prosaposin null mice, suggesting a compensation in LacCer degradation by saposin B for the saposin C deficiency. Skin fibroblasts and tissues from CD-/- mice showed an increase of intracellular prosaposin, impaired prosaposin secretion, deficiencies of saposins C and D and decreases in saposins A and B. In addition, the deficiency of saposin C in CD-/- mice resulted in cellular decreases of acid beta-glucosidase activity and protein. This CD null mouse model provides a tool to explore the in vivo functional interactions of saposins in GSL metabolism and lysosomal storage diseases, and prosaposin's physiological effects.

Functional analysis of 13 GBA mutant alleles identified in Gaucher disease patients: Pathogenic changes and "modifier" polymorphisms.

Gaucher disease, the most prevalent sphingolipidosis, is caused by the deficient activity of acid beta-glucosidase, mainly due to mutations in the GBA gene. Over 200 mutations have been identified worldwide, more than 25 of which were in Spanish patients. In order to demonstrate causality for Gaucher disease, some of them: c.662C>T (p.P182L), c.680A>G (p.N188S), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1093G>A (p.E326K), c.1289C>T (p.P391L), c.1292A>T (p.N392I), c.1322T>C (p.I402T), and the double mutants [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), were expressed in Sf9 cells using a baculovirus expression system. Other well-established Gaucher disease mutations, namely c.1226A>G (p.N370S), c.1342G>C (p.D409H), and c.1448T>C (p.L444P), were also expressed for comparison. The levels of residual acid beta-glucosidase activity of the mutant enzymes produced by the cDNAs carrying alleles c.662C>T (p.P182L), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1289C>T (p.P391L), and c.1292A>T (p.N392I) were negligible. The c.1226A>G (p.N370S), c.1322T>C (p.I402T), c.1342G>C (p.D409H), c.1448T>C (p.L444P), and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]) alleles produced enzymes with levels ranging from 6 to 14% of the wild-type. The three remaining alleles, c.680A>G (p.N188S), c.1093G>A (p.E326K), and [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]), showed higher activity (66.6, 42.7, and 23.2%, respectively). Expression studies revealed that the c.1093G>A (p.E326K) change, which was never found alone in a Gaucher disease-causing allele, when found in a double mutant such as [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), decreases activity compared to the activity found for the other mutation alone. These results suggest that c.1093G>A (p.E326K) should be considered a "modifier variant" rather than a neutral polymorphism, as previously considered. Mutation c.680A>G (p.N188S), which produces a mutant enzyme with the highest level of activity, is probably a very mild mutation or another "modifier variant."

Gaucher disease: pediatric concerns.

Gaucher disease, the most prevalent lysosomal storage disorder, is inherited as an autosomal recessive condition. The gold standard for diagnosis is decreased acid beta-glucosidase activity in the lymphocytes or fibroblasts; molecular analysis of mutations allows for some prognostication of disease severity. Prenatal diagnosis and carrier testing for at-risk families are currently available. There is tremendous phenotypic heterogeneity in the non-neuronopathic form (type I), ranging from clinically asymptomatic to massive hepatomegaly, hypersplenism, growth retardation in children and extensive involvement of bone and lungs. Presence on one allele of the most common mutation, N370S, which is the most prevalent among Ashkenazi Jews for whom there is a predilection for Gaucher disease, is protective of neurological involvement. Some mutations, such as 84GG and IVS2+1, are associated with more severe disease manifestations when appearing as compound heterozygotes with N370S, but when occurring in the homozygous state are not compatible with life. Other mutations, such as L444P, are associated with severe non-neurological disease when occurring as compound heterozygotes with N370S, but when occurring in the homozygous state may be predictive of neurological disease of either acute (type II) or subacute (type III) forms. In the past decade, enzyme replacement therapy has become available which has resulted in a reduction in liver and spleen volume and consequently improved anemia and thrombocytopenia in most patients. It has also engendered catch-up growth in many children, induced improvement in lung involvement secondary to Gaucher disease, and to some extent ameliorated episodes of bone pain. By virtue of treatment, many children who may have been severely affected no longer need to undergo splenectomy to treat hypersplenism, and therefore they are not at risk of bone involvement consequent to the loss of the preferred reservoir for lipid-laden 'Gaucher cells'. However, enzyme treatment is ineffective in reversing neurological signs, requires a lifelong commitment to intravenous infusions, thereby reducing quality of life, and is relatively expensive for many national health schemes. Hence, alternative forms of treatment, such as substrate balance, are being explored. Symptomatic management, including orthopedic surgery, pain relief for bone pain and even splenectomy, still has importance for patients with Gaucher disease. In addition, there is the potential for bone marrow transplantation and, in the future, gene therapy to be curative, particularly for patients with the neuronopathic forms.

Molecular Cloning and Expression of Human Bile Acid β-Glucosidase

A novel microsomal beta-glucosidase was recently purified and characterized from human liver that catalyzes the hydrolysis of bile acid 3-O-glucosides as endogenous compounds. The primary structure of this bile acid beta-glucosidase was deduced by cDNA cloning on the basis of the amino acid sequences of peptides obtained from the purified enzyme by proteinase digestion. The isolated cDNA comprises 3639 base pairs containing 524 nucleotides of 5'-untranslated and 334 nucleotides of 3'-untranslated sequences including the poly(A) tail. The open reading frame predicts a 927-amino acid protein with a calculated M(r) of 104,648 containing one putative transmembrane domain. Data base searches revealed no homology with any known glycosyl hydrolase or other functionally identified protein. The cDNA sequence was found with significant identity in the human chromosome 9 clone RP11-112J3 of the human genome project. The recombinant enzyme was expressed in a tagged form in COS-7 cells where it displayed bile acid beta-glucosidase activity. Northern blot analysis of various human tissues revealed high levels of expression of the bile acid beta-glucosidase mRNA (3.6-kilobase message) in brain, heart, skeletal muscle, kidney, and placenta and lower levels of expression in the liver and other organs.

Gaucher Disease: The Origins of the Ashkenazi Jewish N370S and 84GG Acid β-Glucosidase Mutations

Type 1 Gaucher disease (GD), a non-neuronopathic lysosomal storage disorder, results from the deficient activity of acid beta-glucosidase (GBA). Type 1 disease is panethnic but is more prevalent in individuals of Ashkenazi Jewish (AJ) descent. Of the causative GBA mutations, N370S is particularly frequent in the AJ population, (q approximately .03), whereas the 84GG insertion (q approximately .003) occurs exclusively in the Ashkenazim. To investigate the genetic history of these mutations in the AJ population, short tandem repeat (STR) markers were used to map a 9.3-cM region containing the GBA locus and to genotype 261 AJ N370S chromosomes, 60 European non-Jewish N370S chromosomes, and 62 AJ 84GG chromosomes. A highly conserved haplotype at four markers flanking GBA (PKLR, D1S1595, D1S2721, and D1S2777) was observed on both the AJ chromosomes and the non-Jewish N370S chromosomes, suggesting the occurrence of a founder common to both populations. Of note, the presence of different divergent haplotypes suggested the occurrence of de novo, recurrent N370S mutations. In contrast, a different conserved haplotype at these markers was identified on the 84GG chromosomes, which was unique to the AJ population. On the basis of the linkage disequilibrium (LD) delta values, the non-Jewish European N370S chromosomes had greater haplotype diversity and less LD at the markers flanking the conserved haplotype than did the AJ N370S chromosomes. This finding is consistent with the presence of the N370S mutation in the non-Jewish European population prior to the founding of the AJ population. Coalescence analyses for the N370S and 84GG mutations estimated similar coalescence times, of 48 and 55.5 generations ago, respectively. The results of these studies are consistent with a significant bottleneck occurring in the AJ population during the first millennium, when the population became established in Europe.

Gaucher disease: expression and characterization of mild and severe acid beta-glucosidase mutations in Portuguese type 1 patients.

Type 1 Gaucher disease (GD), the most prevalent lysosomal storage disease, results from the deficient activity of acid beta-glucosidase. Molecular analysis of 12 unrelated Portuguese patients with type 1 GD identified three novel acid beta-glucosidase mutations (F109V, W184R and R395P), as well as three previously reported, but uncharacterized, lesions (R359Q, G377S and N396T). The type 1 probands were either heteroallelic for the well-characterized common lesion, N370S, and the F109V, W184R, R359Q or N396T lesions or homoallelic for the G377S or N396T mutations. Expression of the W184R, R359Q and R395P mutations revealed very low specific activities based on cross-reacting immunologic material (CRIM SAs of 0.0004, 0.016 and 0.045, respectively), consistent with their being found only in type 1 patients who had a neuroprotective N370S allele. In contrast, the F109V, G377S and N396T alleles had significant acid beta-glucosidase activity (CRIM specific activities of 0.15, 0.17, 0.14, respectively), in agreement with their being mild type 1 alleles. Thus, these studies identified additional acid beta-glucosidase mutations in the Portuguese population and demonstrated that the G377S and N396T mutations were neuroprotective, consistent with the mild clinical phenotypes of the type 1 patients who were homoallelic for the G377S and N396T lesions.

Type 1 Gaucher disease presenting with extensive mandibular lytic lesions: Identification and expression of a novel acid ?-glucosidase mutation

The finding of extensive lytic lesions in the mandible of a 19-year-old Ashkenazi Jewish woman led to the diagnosis of Type 1 Gaucher disease. She had extensive skeletal involvement, marked hepatosplenomegaly, and deficient acid beta-glucosidase activity. Mutation analysis identified heteroallelism for acid beta-glucosidase mutations N370S and P401L, the latter being a novel missense mutation in exon 9. Expression of the P401L allele resulted in an enzyme with a reduced catalytic activity (specific activity based on cross-reacting immunological material approximately 0.21), which was similar to that of the mild N370S mutant enzyme. The expression studies predicted a mild phenotype for the proposita's N370S/P401L genotype which was inconsistent with her severe diffuse skeletal disease and organ involvement. Since lytic mandibular lesions may be complicated by osteomyelitis, pathologic fracture, and tooth loss, regular dental assessments in Type 1 Gaucher patients should be performed.

12 A practical approach to diagnosis and management of Gaucher's disease

The diagnosis of Gaucher's disease is established by demonstration of reduced acid beta-glucosidase activity in peripheral blood leukocytes. Genotyping at the glucocerebrosidase gene locus can give additional prognostic information and facilitate carrier detection. However, extreme phenotypic diversity precludes reliable prediction of prognosis in individual patients. Histological diagnosis of Gaucher's disease is unnecessary and can be misleading. A range of clinical, radiological and laboratory parameters are useful for staging disease activity which is central to achieving optimal timing to initiate enzyme therapy. Treatment should be individualized to obtain maximum therapeutic response. The recent introduction of chitotriosidase measurements has provided a valuable indicator of total cellular burden of storage cells. Serial measurements of chitotriosidase activity are useful for monitoring disease progression as well as response to therapy. A number of adjuvant therapies are available for use in conjunction with enzyme treatment. Special considerations apply to management of affected children.

Therapeutic delivery of proteins to macrophages: implications for treatment of Gaucher's disease

The primary defect in Gaucher's disease, a lysosomal disorder affecting macrophages, is in the activity of glucocerebrosidase. Treatment with exogenous enzyme (modified to increase its affinity for macrophage glycoprotein receptors) aims to restore this activity. However, the fate of the exogenous enzyme in vivo is unknown. We used radiolabelled enzyme to assess macrophage receptor activity for mannosylated ligands in vivo. We examined the uptake and tissue distribution of radiolabelled enzyme molecules by gamma scintigraphy after bolus injection of iodine-123-labelled recombinant or placental enzyme (imiglucerase and alglucerase, respectively) in eight patients with type 1 Gaucher's disease, and in one healthy individual. The metabolism of the tracer enzyme was followed by scintigraphy and by analysis of blood, urine, and faeces. The tracer enzyme was rapidly cleared from blood (half-life 4.7 min [SD 1.0]). Concomitantly, there was avid uptake by the liver (about 30% of the injected dose), the spleen (about 15%), and the bone marrow. 40-55% of the tracer was cleared rapidly from the viscera (half-life 1-2 h) and 45-60% was cleared slowly (half-life 34-42 h). The half-life in the bone marrow was 14.1 h. Infusion of alglucerase at dose of 5 U/kg bodyweight normalised acid beta-glucosidase activity of splenic Gaucher's cells in vivo. When the enzyme was administered at a seven-fold higher dose (35 U/kg over 1 h), the receptor-mediated uptake in vivo was saturated, as shown by the increase in blood-clearance half-life of tracer enzyme from 4.5 min to 12 min. Avid and saturable uptake of modified glucocerebrosidase was found, which indicates high-affinity targeting to the macrophage system in vivo. The rate of enzyme turnover suggests a rational basis for use of this therapy in treatment of Gaucher's disease.

Functional Organization of Saposin C: DEFINITION OF THE NEUROTROPHIC AND ACID β-GLUCOSIDASE ACTIVATION REGIONS

Saposin C is an essential co-factor for the hydrolysis of glucosylceramide by acid beta-glucosidase in mammals. In addition, prosaposin promotes neurite outgrowth in vitro via sequences in saposin C. The regional organization of these neurotrophic and activation properties of saposin C was elucidated using recombinant or chemically synthesized saposin Cs from various regions of the molecule. Unreduced and reduced proteins were analyzed by electrospray-mass spectrometry to establish the complement of disulfide bonds in selected saposin Cs. Using saposin B as a unreactive backbone, chimeric saposins containing various length segments of saposin B and C localized the neurotrophic and acid beta-glucosidase activation properties to the carboxyl- and NH2-terminal 50% of saposin C, respectively. The peptide spanning residues 22-31 had neurotrophic effects. Molecular modeling and site-directed mutagenesis localized the activation properties of saposin C to the region spanning residues 47-62. Secondary structure was needed for retention of this property. Single substitutions of R and S at the conserved cysteines at 47 or 78 diminished but did not obliterate the activation properties. These results indicate the segregation of neurotrophic and activation properties of saposin C to two different faces of the molecule and suggest a topographic sequestration of the activation region of prosaposin for protection of the cell from adverse hydrolytic activity of acid beta-glucosidase.

Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid beta-glucosidase

Small (80-amino acid) glycoproteins or saposins are important for the in vivo function of several lysosomal hydrolases. Four saposins, A, B, C, and D, are encoded by a single locus termed prosaposin. Saposins C and A are thought to function in vivo as activators of acid beta-glucosidase. The physiologic role of saposin C has been confirmed, whereas that of saposin A role has not. To investigate the effects of saposins C and A on acid beta-glucosidase activity, the coding sequence for the individual saposins was expressed in Escherichia coli and the recombinant proteins purified to homogeneity. Recombinant and natural saposins A and C activated acid beta-glucosidase similarly only in micromolar amounts. Saposin C had specific activation of acid beta-glucosidase activity at < 200 nM. A second phase of activation was achieved at > 1 microM. In comparison, saposin A consistently activated acid beta-glucosidase only at > 1 microM. Two mutant saposins C (Cys382-->Phe and Cys382--Gly) were created and shown to compete with saposin C for a site on acid beta-glucosidase. The mutant saposins did not activate the enzyme. Recombinant saposin A (< 200 nM) competed with saposin C for a site on the enzyme but without activating effects. These studies show that saposin A is not an in vitro activator of acid beta-glucosidase at physiologic concentrations, although binding occurs without activating acid beta-glucosidase. The studies with mutant saposins C indicate that the binding and activation effects of saposins C are distinct events. These results indicate that the saposin C-induced conformational change in the enzyme occurs via highly specific, probably multivalent, interactions between acid beta-glucosidase and saposin C.

Glucosylceramide and Glucosylsphingosine Metabolism in Cultured Fibroblasts Deficient in Acid β-Glucosidase Activity1

The metabolism of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) was studied using cultured fibroblasts deficient in acid beta-glucosidase activity. In fibroblasts from patients with Gaucher's disease, in vitro beta-glucosidase activities were 2.7-11.7% and 4.8-13.6% of control values when 4-methylumbelliferyl beta-D-glucoside and GlcSph were used as substrates, respectively. In spite of the enzyme deficiency, GlcCer and GlcSph, the natural substrates of the deficient enzyme, did not accumulate in the cells. When normal fibroblasts were incubated with conduritol B epoxide (CBE), a specific inhibitor of acid beta-glucosidase, the in vitro enzyme activities decreased dose-dependently (2.2-2.4% of control values at 50 microM CBE), and GlcCer and GlcSph accumulated in the cells at concentrations of CBE higher than 50 microM. To investigate the intracellular metabolism of GlcCer and GlcSph, either radioactive GlcCer or GlcSph was loaded onto cultured fibroblasts. In fibroblasts treated with a high dose of CBE (1 mM), the degradation of GlcCer and GlcSph was retarded (5-21% on day 7; normal range, 81-99%), while in fibroblasts from patients with Gaucher's disease, both the pattern and rate of the degradation of the lipids (83-97% on day 7) were almost the same as those seen in the control cells. These results indicate that in Gaucher's disease fibroblasts the intracellular metabolism of GlcCer and GlcSph is normal in spite of the deficiency in beta-glucosidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Augmentation in Moderate to Life-Threatening Gaucher Disease

Gaucher disease type 1 (GD type 1) is the most prevalent lysosomal storage disease and has its highest frequency in the Ashkenazi Jewish population. Deficiency of the enzyme, acid beta-glucosidase, results in the deposition of glucocerebroside primarily in macrophages. The accumulation of such "Gaucher cells" leads to visceromegaly, hepatic and bone marrow dysfunction, hypersplenism, and bony disease. Eleven GD type 1 patients, ages 4-52 y, with moderate to life-threatening manifestations, received 6-12 mo of enzyme augmentation with a macrophage-targeted acid beta-glucosidase preparation. Within 6 mo, substantial increases in Hb levels (mean = +30%) and platelet counts (mean = +39%) were observed. Hepatic and splenic volumes decreased by approximately 20% (range = 3-35%) and approximately 35% (20-52%), respectively. Hematologic and hepatic volume improvements were similar in the splenectomized (n = 4) and nonsplenectomized (n = 7) patient groups. In this patient population, no major differences were observed in the hematologic and visceral improvements with enzyme doses of 30, 50, or 60 IU/kg administered every 2 wk. Normal levels of acid beta-glucosidase activity were present in hepatic autopsy samples from one patient 11 d after enzyme infusion. In comparison, exogenous activity was absent from brain and lung specimens of the same patient. High levels (approximately 10-fold normal) were present in bone marrow samples from two patients obtained at 1 and 11 d after infusions. These studies demonstrate biochemical and clinical improvements by targeted enzyme augmentation in GD type 1, even in far advanced, life-threatening involvement.(ABSTRACT TRUNCATED AT 250 WORDS)

Marked variation in blood beta-hexosaminidase in Gaucher disease

Gaucher disease is due to a primary deficiency of acid beta-glucosidase activity and is associated with secondary elevations of several plasma/serum lysosomal enzyme activities, including beta-hexosaminidase. We analyzed plasma and serum beta-hexosaminidase A & B activities in 55 patients with enzyme-documented Gaucher disease. The mean beta-hexosaminidase activity was increased and the percent of the A isozyme decreased, consistent with earlier studies. Gaucher disease patients had 2,067 ± 1,491 nmol ml −1 h −1 units of beta-hexosaminidase activity with 51.9 ± 15.5% beta-hexosaminidase A compared to 1,086 ± 260 nmol ml −1 h −1 and 67.8 ± 4.0% beta-hexosaminidase A in normal controls and 965 ± 261 nmol ml −1 h −1 and 43.6 ± 5.5% beta-hexosaminidase A in Tay-Sachs disease heterozygotes. Contrary to previous reports, marked heterogeneity of both total plasma/serum enzyme activity and isozyme pattern was noted, as some patients had normal enzyme levels and others had severe reductions in the percent of hexosaminidase A. These data argue against the suggestions of recent studies that routine serum beta-hexosaminidase testing done in Tay-Sachs disease heterozygote detection programs can be effectively used to screen for patients with Gaucher disease.

A 27-Year experience with splenectomy for Gaucher's disease

Gaucher's disease is an inherited metabolic disorder caused by the defective activity of acid beta-glucosidase and the resultant accumulation of glucosyl ceramide-laden macrophages in the liver, bone, and spleen. Splenectomy is the preferred treatment for patients with Gaucher's disease who develop massive splenomegaly with accompanying hypersplenism and/or mechanical pressure symptoms. The charts of 48 patients with Gaucher's disease undergoing splenectomy at our institution between January 1963 and December 1989 were analyzed to determine the short- and long-term results of this procedure. Thirty-five (73%) patients had total splenectomy, whereas 13 (27%) patients had partial splenectomy. There was one postoperative death (after total splenectomy), and 13 patients (27%) had postoperative complications. Eleven patients (23%) presented with accelerated bone disease after total splenectomy (mean follow-up: 96 months). No patients having partial splenectomy (mean follow-up: 25 months) developed progressive bone disease. Eight patients have died since surgery. All four deaths due to malignant disease occurred in patients after total splenectomy. The results of this largest-ever reported series of splenectomy for Gaucher's disease confirm that while either total or partial splenectomy can be performed with minimal morbidity and mortality, total splenectomy is accompanied by more aggressive bone disease and a predisposition to malignancy. Prospective, randomized trials are needed to substantiate whether partial splenectomy is indeed the treatment of choice for splenomegaly associated with Gaucher's disease.

Analyses of catalytic activity and inhibitor binding of human acid beta-glucosidase by site-directed mutagenesis. Identification of residues critical to catalysis and evidence for causality of two Ashkenazi Jewish Gaucher disease type 1 mutations.

Analyses of catalytic properties and inhibitor binding were conducted to investigate the molecular basis of active site function of human acid beta-glucosidases (EC 3.2.1.45) expressed from normal and Gaucher disease Type 1 alleles. Comparative studies were conducted with enzymes expressed from natural (spleen and fibroblasts) alleles or from mutagenized cDNAs in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system. Mutant cDNAs containing Thr43 to Lys43 (beta-GlcThr43----Lys) and Asp358 to Glu358 (beta-GlcAsp358----Glu) substitutions and two cDNAs containing Ashkenazi Jewish Gaucher disease Type 1 mutations, Arg120 to Gln120 (beta-GlcArg120----Gln) and Asn370 to Ser370 (beta-GlcAsn370----Ser) were expressed and the gene products characterized by enzymatic, immunologic, and inhibitor studies. Genotypes at the acid beta-glucosidase locus in selected Gaucher disease Type 1 patients were determined by allele-specific oligonucleotide hybridization of amplified genomic DNA. Compared with normal, recombinant or natural enzymes expressed from beta-GlcAsn370----Ser alleles had about 2-5-fold decreased specific activity based on CRIM (cross-reacting immunologic material). The beta-GlcArg120----Gln cDNA expressed catalytically inactive CRIM in Sf9; consistent with the 9-fold decreased CRIM-specific activity of the natural enzyme from a beta-GlcArg120----Gln/beta-GlcAsn370----Ser genetic compound. The beta-GlcAsp358----Glu cDNA expressed catalytically inactive CRIM in Sf9 cells. The presence of natural or recombinant enzyme expressed from beta-GlcAsn370----Ser alleles was sufficient to confer 3-5-fold increased IC50 values for deoxynojirimycin, glucosylsphingosine, and N-alkyl-glucosylamine derivatives. Progress curves for inhibition by the slow-tight binding N-alkyl-glucosylamines indicated that the beta-Glc-Asn370----Ser mutation did not alter a conformational change induced by these reaction intermediate analogues. These results provide evidence that the beta-GlcArg120----Gln and beta-GlcAsn370----Ser mutations found in Gaucher disease Type 1 patient genomes are the molecular bases of the enzymatic dysfunction. In addition, the region including Arg120 and that encompassing Asp358 and Asn370 contain residues critical to active site formation or participation in the catalytic mechanism.

Gaucher disease type 1: cloning and characterization of a cDNA encoding acid beta-glucosidase from an Ashkenazi Jewish patient.

Gaucher disease (GD) type 1 is the most prevalent lysosomal storage disease and the most prevalent genetic disease among the Ashkenazi Jews. The defective activity of acid beta-glucosidase is the enzymatic basis of GD and is inherited as an autosomal recessive trait. To investigate the genetic basis of Ashkenazi Jewish GD type 1, a cDNA encoding acid beta-glucosidase was isolated from a cDNA library constructed using splenic poly(A)+RNA from a patient. The cDNA was sequenced to identify mutations, and the presence of a single missense mutation in the patients' genome was confirmed by selective oligonucleotide hybridization and by restriction endonuclease digestion analyses of amplified genomic sequences. This G----A transition (Arg-119 to Gln-119) was present heterozygously in the index patient and his affected third cousin but was not present in normal non-Jewish individuals. This mutation is the second single base mutation found in Ashkenazi Jewish GD type 1 patients. Furthermore, results obtained with the affected third cousin suggest that at least three mutant alleles may be present in this GD subpopulation.

Effect of immune complexes, acting on the heart, on cardio- and hemodynamics in dogs

Dextran blue decreases the activity of lysosomal acid cholesteryl esterase of rat liver at a concentration from 0.25 M to 10 M without altering acid phosphatase, acid beta-galactosidase and beta-glucosidase activities. The dextran blue filled lysosomes with a high degree of purity prepared by centrifugation over the linear sucrose density gradient contained insignificant impurities (up to 19%) of protein from other organelles. The specific activity of acid phosphatase, beta-galactosidase and beta-glucosidase was increased 35-40-fold in this fraction, whereas the activity of acid cholesteryl esterase rose but 14.7-fold. Chromatography on a Sepharose 2B column of the digitonin-digested native and dextran-containing lysosomes attests to the formation of large dextran aggregates with lysosomal matrix proteins. Since aggregation of dextran blue with acid phosphatase, beta-galactosidase and beta-glucosidase does not affect their activities, it is concluded that to bring about hydrolysis of lipoprotein cholesterol esters, it is necessary that cholesteryl esterase be associated with hydrophobic macromolecules. Moreover, dextran blue can be used for simulation cholesterol esters deposition in lysosomes.