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Biomedical subjects

G A Grabowski

Publications and source records attributed to G A Grabowski.

At least 73 records · Page 4Linked to original sources
PubMed1993

Human acid beta-glucosidase. N-glycosylation site occupancy and the effect of glycosylation on enzymatic activity.

The five potential N-glycosylation sites (sequons) of human acid beta-glucosidase were individually mutated to determine site occupancy and the effect of site occupancy on selected catalytic and stability properties of this enzyme. Each N-glycosylation consensus sequence [Asn-Xaa-(Ser/Thr)] was obliterated by individually substituting glutamine (Q) for asparagine (N). By expression of the normal and mutated cDNAs in insect (Sf9) and COS-1 cells and subsequent immunoblotting with anti-human acid beta-glucosidase antibodies, the four sequons at Asn-19, Asn-59, Asn-146, and Asn-270 were shown to be glycosylated in either source. The sequon at Asn-462 was never occupied. The mutant enzymes N59Q, N146Q, and N270Q were catalytically active and had normal interactions with active site-directed inhibitors as well as with the activators, phosphatidylserine and saposin C. Of the occupied sequons, N-glycosylation of the first was critical to the synthesis of a catalytically active enzyme. Alteration of this sequon, Asn-19-Ala-20-Thr-21, by the substitutions N19Q, N19D, N19E, or T21G led to a lack of glycosylation at this site. Enzymes containing N19Q, N19E, or T21G had significant decreases (3- to 60-fold) in intrinsic enzyme activity. The N19D enzyme had nearly normal catalytic activity and had enhanced activation by phosphatidylserine. These results show that sequon occupancy as well as steric effects at residue 19 are important for the development of an active conformer of this enzyme. This is the first example of a lysosomal hydrolase that requires sequon occupancy for the synthesis of a catalytically active enzyme.

Amino Acid Sequence↗
PubMed1993

Enzyme therapy in Gaucher disease type 1: dosage efficacy and adverse effects in 33 patients treated for 6 to 24 months.

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent genetic disease among the Ashkenazi Jews (q approximately 0.047). The disease results from inherited defects of acid beta-glucosidase and the accumulation of the substrate, glucosylceramide, in cells of monocyte/macrophage origin. The therapeutic response to macrophage-targeted (alpha-mannosyl-terminated) alglucerase (Ceredase, at 60 to 15 IU/kg every 2 weeks) was analyzed in 33 patients (age range, 2 to 63 years; 15 splenectomized) with extensive Gaucher disease over periods of 6 to 24 months. The efficacy of several different doses and dosage reductions was evaluated. In patients with anemia (n = 30) and/or thrombocytopenia (n = 19), hemoglobin levels and platelet counts increased by 0% to 178% and 15% to 155%, respectively, within 3 to 12 months. In patients with splenomegaly (n = 17) and/or hepatomegaly (n = 28), liver and spleen volumes decreased in 6 months from 7% to 64% and 8% to 84% by 12 months, respectively. Hematologic and visceral improvements were noted at any doses between 60 and 15 IU/kg every 2 weeks. Furthermore, these positive initial therapeutic responses were persistent throughout therapy, with doses reduced by 50%. Pulmonary Gaucher disease did not improve clinically in 3 patients. Unrelated cirrhotic (n = 2), cholestatic (n = 1), or renal disease (n = 1) did not influence the rate of patient improvement. Two of five patients who developed serum antibodies against alglucerase during the first 6 to 12 months of therapy had mild antibody reactions. This study shows similar regression of clinical Gaucher disease manifestations with enzyme therapy, using doses between 30 and 60 IU/kg every 2 weeks. Therapeutic efficacy was not diminished after 50% to 75% dose reductions or in the presence of anti-enzyme antibodies.

Acid Phosphatase↗
PubMed1993

The role of neurogenetics in Gaucher disease.

Gaucher disease is the most prevalent hereditary metabolic storage disorder, and the most common genetic disease in individuals of Ashkenazic Jewish ancestry. Patients with Gaucher disease have been classified into three clinical phenotypes. Patients with type 1 disease exhibit markedly variable hepatosplenomegaly, anemia, thrombocytopenia, skeletal, and, to a lesser extent, pulmonary and kidney involvement. The central nervous system does not appear to be involved. In patients with type 2 Gaucher disease, hepatosplenomegaly and extensive central nervous system damage are apparent in infancy. These patients usually die between 1 and 2 years of age. Patients with type 3 Gaucher disease have been subclassified into types 3a and 3b. Type 3a patients exhibit mild-to-moderate hepatosplenomegaly and slowly progressive neurologic deterioration. Recurrent myoclonic seizures are common. Patients with type 3b Gaucher disease exhibit splenomegaly along with extensive hepatomegaly that is frequently accompanied by esophageal varices. Horizontal supranuclear gaze paresis is the major neurologic sign. Excessive quantities of glucocerebroside accumulate in the organs of patients with Gaucher disease because of a deficiency of the enzyme glucocerebrosidase. In the vast majority of patients, the reduction of glucocerebrosidase activity is caused by mutations in the gene that codes for glucocerebrosidase. In a few instances, glucocerebroside accumulates due to a lack of saposin C, a cohydrolase that is required in addition to glucocerebrosidase for the catabolism of glucocerebroside. Mutations in the glucocerebrosidase gene are discussed in the context of the severity of disease and the presence or absence of nervous system involvement. Enzyme replacement therapy is highly beneficial for patients with type 1 Gaucher disease. Enzyme replacement is also being investigated for patients with type 3b Gaucher disease. Novel procedures must be developed to deliver glucocerebrosidase to the nervous system so that patients with type 2 and type 3a Gaucher disease can be helped. Exploration of gene therapy for Gaucher disease is under way.

Alleles↗
PubMed1993

Phenotype/genotype correlations in Gaucher disease type I: clinical and therapeutic implications.

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent genetic disease among Ashkenazi Jews. Gaucher disease type 1 is characterized by marked variability of the phenotype and by the absence of neuronopathic involvement. To test the hypothesis that this phenotypic variability was due to genetic compounds of several different mutant alleles, 161 symptomatic patients with Gaucher disease type 1 (> 90% Ashkenazi Jewish) were analyzed for clinical involvement, and their genotypes were determined. Qualitative and quantitative measures of disease involvement included age at onset of the disease manifestations, hepatic and splenic volumes, age at splenectomy, and severity of bony disease. Highly statistically significant differences (P < .005) were found in each clinical parameter in patients with the N370S/N370S genotype compared with those patients with the N370S/84GG, N370S/L444P, and N370S/? genotypes. The symptomatic N370S homozygotes had onset of their disease two to three decades later than patients with the other genotypes. In addition, patients with the latter genotypes have much more severely involved livers, spleens, and bones and had a higher incidence of splenectomy at an earlier age. These predictive genotype analyses provide the basis for genetic care delivery and therapeutic recommendations in patients affected with Gaucher disease type 1.

Adolescent↗
PubMed1992

Structure and evolution of the human prosaposin chromosomal gene.

The gene for prosaposin was characterized by sequence analysis of chromosomal DNA to gain insight into the evolution of this locus that encodes four highly conserved sphingolipid activator proteins or saposins. The 13 exons ranged in size from 57 to 1200 bp, while the introns were from 91 to 3812 bp in length. The regions encoding saposins A, B, and D each had three exons, while that for saposin C had only two. This sequence included the regions that encode the carboxy terminus of the signal peptide, the four mature prosaposin proteins, and the 3' untranslated region. Primer extension studies indicated that over 99% of the coding sequence was contained in these 19,985 bp. Use of PCR and reverse PCR techniques indicated that the most 5' coding approximately 140 bp contained large introns and at least two small exons. Analyses of the intronic positions in the saposin regions indicated that this gene evolved from an ancestral gene by two duplication events and at least one gene rearrangement involving a double crossover after introns had been inserted into the gene.

Animals↗
PubMed1992

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)

Adolescent↗
PubMed1992

Allogenic bone marrow transplantation in severe Gaucher disease.

Gaucher disease is the most prevalent lysosomal storage disease. This autosomal recessive disease is caused by the defective activity of the enzyme acid beta-glucosidase and the resultant accumulation of glucosylceramide primarily within cells of the reticuloendothelial system. Because the primary manifestations of Gaucher disease are due to involvement of monocyte/macrophage-derived cells, this disease is thought to be an excellent candidate for curative intervention via bone marrow transplantation (BMT). A Hispanic female with subacute neuronopathic Gaucher disease and rapidly progressing visceral manifestations underwent BMT at 23 mo of age using her histocompatible normal brother as the donor. Cytogenetic analyses demonstrated complete, stable engraftment by 1 mo post-BMT. During the subsequent 24 mo, clinical, biochemical, enzymatic, and histologic studies demonstrated nearly complete correction in the viscera. Her neuropathic manifestations did not progress. Complete reconstitution of enzymatic activity in peripheral blood leukocytes was achieved by 1 mo. Cytogenetic analyses demonstrated complete engraftment by d 79 and nearly complete loss of bone marrow Gaucher cells was observed by 8 mo. Plasma glucosylceramide levels normalized by 8-12 mo. Nearly coincident improvements in hepatic size, enzyme levels, and histology were found by 12-24 mo post-BMT. Fatal sepsis occurred at 24 mo post-BMT. Autopsy revealed sparse Gaucher cells in clusters in the liver, lymph nodes, and lungs as well as the lack of periadventitial Gaucher cells surrounding brain vessels. The findings provide the time course and rationale for studies directed to gene therapy via BMT for this disease after introduction of acid beta-glucosidase gene constructs into autologous pluripotent stem cells of selected Gaucher disease patients.

Bone Marrow Transplantation↗
PubMed1992

Gaucher disease: A G+1----A+1 IVS2 splice donor site mutation causing exon 2 skipping in the acid beta-glucosidase mRNA.

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent Jewish genetic disease. About 30 identified missense mutations are causal to the defective activity of acid beta-glucosidase in this disease. cDNAs were characterized from a moderately affected 9-year-old Ashkenazi Jewish Gaucher disease type 1 patient whose 80-year-old, enzyme-deficient, 1226G (Asn370----Ser [N370S]) homozygous grandfather was nearly asymptomatic. Sequence analyses revealed four populations of cDNAs with either the 1226G mutation, an exact exon 2 (delta EX2) deletion, a deletion of exon 2 and the first 115 bp of exon 3 (delta EX2-3), or a completely normal sequence. About 50% of the cDNAs were the delta EX2, the delta EX2-3, and the normal cDNAs, in a ratio of 6:3:1. Specific amplification and characterization of exon 2 and 5' and 3' intronic flanking sequences from the structural gene demonstrated clones with either the normal sequence or with a G+1----A+1 transition at the exon 2/intron 2 boundary. This mutation destroyed the splice donor consensus site (U1 binding site) for mRNA processing. This transition also was present at the corresponding exon/intron boundary of the highly homologous pseudogene. This new mutation, termed "IVS2 G+1----A+1," is the first splicing mutation described in Gaucher disease and accounted for about 3.4% of the Gaucher disease alleles in the Ashkenazi Jewish population. The occurrence of this "pseudogene"-type mutation in the structural gene indicates the role of acid beta-glucosidase pseudogene and structural gene rearrangements in the pathogenesis of this disease.

Aged↗
PubMed1991

Human acid beta-glucosidase. Use of inhibitory and activating monoclonal antibodies to investigate the enzyme's catalytic mechanism and saposin A and C binding sites.

Of 14 identified epitopes on human GCase (acid beta-glucosidase), monoclonal antibodies (MCABs) recognizing 3 produced inhibition and 1 resulted in activation of GCase. MCABs F1 and F2 completely, and MCAB 61 partially (approximately 70%), inhibited GCase activity. Substrates and active site-directed inhibitors (specific sphingolipid and 5-amino-5-deoxyglucose derivatives) protected the enzyme from inhibition by MCAB F1 and F2, but not that by MCAB 61. Conduritol B epoxide did not protect GCase from the inhibition by these MCABs when covalently bound to the active site. These results indicated highly specific binding requirements of MCABs F1 and F2 for residues in a complex active site. In comparison, kinetic analyses using GCase transition state analogues, N-alkyl-glucosylamines, and MCAB 61 demonstrated that this MCAB "freezes" the conformation of the enzyme and inhibits GCase by preventing formation of a conformer needed for maximal catalytic rates. The activating MCAB 122 mimicked the effects of saposin C and competed with this natural activator for residues on the enzyme. Interaction of saposin A and saposin C or MCAB 122 with GCase produced a synergistic effect leading to a marked sensitization of the enzyme to these activators. No such synergism or additivity was found for the maximal catalytic rate since it could be achieved by saturating amounts of any one or combinations of these activators. In the presence of MCAB 61, only 15 to 25% of the maximal activation of GCase was obtained by saposin C or MCAB 122, indicating that the major activation effects of these effectors derived from an induction of a GCase conformational change. These results demonstrate that saposins A and C mediate their activating effects by binding to distinct sites on GCase. Furthermore, major components of the mechanisms for catalysis and saposin C activation are due to conformational changes during the transition state. These findings have implications for understanding the perturbations of GCase function due to the missense mutations which cause Gaucher disease.

Antibodies, Monoclonal↗
PubMed1991

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.

Adult↗
PubMed1991

High frequency of the Gaucher disease mutation at nucleotide 1226 among Ashkenazi Jews.

Reliable estimates of the frequency of Gaucher disease-producing mutations are not available. The high frequency of Gaucher disease in the Ashkenazi Jewish population is due to the occurrence of a mutation at nucleotide (nt) 1226. We have screened 593 DNA samples from normal Ashkenazi Jews, as well as 62 DNA samples from all our Ashkenazi Jewish patients with Gaucher disease, for the presence of the 1226 mutation. In the 593 presumed normal Ashkenazi Jewish individuals the 1226 mutation was identified in the heterozygous state in 37 and in the homozygous state in two, giving a gene frequency of .035 for the mutation. This 1226 mutation represented 73% of the 124 Gaucher disease alleles in Jewish Gaucher disease patients. Accordingly we estimate that the gene frequency for Gaucher disease among the Ashkenazi Jewish population is .047, which is equivalent to a carrier frequency of 8.9% and a birth incidence of 1:450.

Adolescent↗
PubMed1991

Gaucher disease: heterologous expression of two alleles associated with neuronopathic phenotypes.

To investigate the molecular basis for the distinct neuronopathic phenotypes of Gaucher disease, acid beta-glucosidases expressed from mutant DNAs in Gaucher disease type 2 (acute) and type 3 (subacute) patients were characterized in fibroblasts and with the baculovirus expression system in insect cells. Expression of the mutant DNA encoding a proline-for-leucine substitution at amino acid 444 (L444P) resulted in a catalytically defective, unstable acid beta-glucosidase in either fibroblasts from L444P/L444P homozygotes or in insect cells. This mutation was found to be homoallelic in subacute neuronopathic (type 3) Gaucher disease. In comparison, expression of the mutant cDNA encoding an arginine-for-proline substitution at amino acid 415 (P415R) resulted in an inactive and unstable protein in insect cells. This allele was found only in a type 2 patient with the L444P/P415R genotype. The substantial variation in the type 3 phenotype (L444P homozygotes) suggests the complex nature of the molecular basis of phenotypic variation in Gaucher disease. Yet, the association of neuronopathic phenotypes with alleles producing severely compromised (L444P) or functionally null (P415R) enzymes indicates that the effective level of residual activity at the lysosome is likely to be a major determinant of the severity of Gaucher disease.

Acute Disease↗
PubMed1990

Human acid beta-glucosidase: use of sphingosyl and N-alkyl-glucosylamine inhibitors to investigate the properties of the active site.

Human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the beta-glucosidic bonds of glucosylceramide and synthetic beta-glucosides. The specificity of binding to the active site of this enzyme was evaluated using series of inhibitors including synthetic sphingosines, N-alkyl(Cn)-deoxynojirimycins (1,5-dideoxy-5-iminoglucose) and N-Cn-glucosylamines. The sphingosines were rapidly reversible inhibitors with maximal potency (IC50 approximately 78-150 micro M) at chain lengths of 14-18 carbons. The presence of unsaturation between C4 and C5 was required for inhibition of enzyme activity. Neither the nature of this bond (double or triple bond) nor the presence of erythro or threo configurations at C2 influenced inhibitory potency. The N-C10- to N-C14-deoxynojirimycins were rapidly reversible inhibitors with Ki approximately 8.5 nM. In comparison, the 1-amino glucose derivatives, i.e., N-Cn-glucosylamines (n = 12-18), were more potent (IC50 approximatley 0.3-3 nM) and their maximal inhibitory potencies were dependent on time as well as enzyme and substrate concentrations: i.e., the N-C12- to N-C18-glucosylamines were competitive, slow-tight binding inhibitors. Analyses of progress curves at various N-Cn-glucosylamine (n = 14-18) concentrations indicated the formation of rapidly dissociating initial EI collison complex which then undergoes a conformational change to a slowly reversible EI complex. These results were consistent with the long chain N-Cn-glucosylamines being reaction intermediate analogues and with this enzyme's hydrolytic mechanism requiring a conformational change during the transition state.

1-Deoxynojirimycin↗
PubMed1990

Human acid beta-glucosidase: glycosylation is required for catalytic activity.

The role of oligosaccharide modification in human acid beta-glucosidase function was investigated. This lysosomal enzyme has five putative N-glycosylation sites, four of which are occupied. The unglycosylated human protein was stable when expressed in bacteria or in Spodoptera frugiperda cells in the presence of tunicamycin but lacked catalytic activity. Deglycosylation of purified acid beta-glucosidase from human placenta with N-Glycanase under native conditions resulted in the removal of an accessible oligosaccharide chain from a single site with no effect on activity, whereas complete deglycosylation resulted in proportionate loss of activity. These studies demonstrate that occupancy of at least one glycosylation site is required for the formation and maintenance of acid beta-glucosidase in an active conformation.

Animals↗
PubMed1990

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.

Amino Acid Sequence↗