Two novel (1098insA and Y313H) and one rare (R359Q) mutations detected in exon 8 of the beta-glucocerebrosidase gene in Gaucher's disease patients.
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Base Sequence↗
Biomedical subjects
R Gonzàlez-Duarte
Publications and source records attributed to R Gonzàlez-Duarte.
Autosomal recessive retinitis pigmentosa in Spain: evaluation of four genes and two loci involved in the disease.
Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous form of retinal degeneration. The genes for the beta-subunit of rod phosphodiesterase (PDEB), rhodopsin (RHO), peripherin/RDS (RDS) and the rod outer segment membrane protein 1 (ROM1), as well as loci at 6p and 1q, have previously been reported as the cause of ARRP. In order to determine whether they are responsible for the disease in Spanish pedigrees, linkage and homozygosity studies using markers at these loci were carried out on 47 Spanish ARRP families. SSCP analysis was performed to search for mutations in the genes cosegregating with the disease in particular pedigrees. Three homozygous mutations in the PDEB gene were found, thus accounting for 6% of the cases. No other disease-causing mutation was observed in the other genes analysed, nor was significant evidence found for the involvement of the loci at 6p or 1q. On the basis of these data, it is unlikely that these genes and loci account for a considerable proportion of ARRP cases.
Calcium-Binding Proteins↗
Involvement of the C-terminal tail in the activity of Drosophila alcohol dehydrogenase. Evaluation of truncated proteins constructed by site-directed mutagenesis.
Drosophila alcohol dehydrogenase belongs to the heterogeneous family of short-chain dehydrogenases/reductases, which does not include the well characterized mammalian alcohol dehydrogenases. Although it is clear that the main biological role of this enzyme is in alcohol oxidation, in the absence of the three-dimensional conformation only partial information on the protein regions involved in the active site, and the coenzyme and substrate interacting cavities is available. Two segments have already been identified, a coenzyme-binding segment at the N-terminus, and the reactive Tyr152 and Lys156 residues. Limited proteolytic assays had suggested the involvement of the 13 C-terminal amino acids in the function of the enzyme. By site-directed mutagenesis, we have constructed eight different truncated mutant enzymes and expressed them in Escherichia coli. The purified mutant enzymes have been recovered and characterized using monoclonal antibodies. Kinetic analysis and stability assays have been performed, and clearly demonstrate the contribution of the last 13 amino acids to the activity. We hypothesize that the C-terminal tail constitutes an essential region for maintaining the hydrophobicity of the catalytic pocket needed for binding of the substrate.
Alcohol Dehydrogenase↗
Short-chain dehydrogenases/reductases (SDR).
Short-chain dehydrogenases/reductases (SDR) constitute a large protein family. Presently, at least 57 characterized, highly different enzymes belong to this family and typically exhibit residue identities only at the 15-30% level, indicating early duplicatory origins and extensive divergence. In addition, another family of 22 enzymes with extended protein chains exhibits part-chain SDR relationships and represents enzymes of no less than three EC classes. Furthermore, subforms and species variants are known of both families. In the combined SDR superfamily, only one residue is strictly conserved and ascribed a crucial enzymatic function (Tyr 151 in the numbering system of human NAD(+)-linked prostaglandin dehydrogenase). Such a function for this Tyr residue in SDR enzymes in general is supported also by chemical modifications, site-directed mutagenesis, and an active site position in those tertiary structures that have been characterized. A lysine residue four residues downstream is also largely conserved. A model for catalysis is available on the basis of these two residues. Binding of the coenzyme, NAD(H) or NADP(H), is in the N-terminal part of the molecules, where a common GlyXXXGlyXGly pattern occurs. Two SDR enzymes established by X-ray crystallography show a one-domain subunit with seven to eight beta-strands. Conformational patterns are highly similar, except for variations in the C-terminal parts. Additional structures occur in the family with extended chains. Some of the SDR molecules are known under more than one name, and one of the enzymes has been shown to be susceptible to native, chemical modification, producing reduced Schiff base adducts with pyruvate and other metabolic keto derivatives. Most SDR enzymes are dimers and tetramers. In those analyzed, the area of major subunit contacts involves two long alpha-helices (alpha E, alpha F) in similar and apparently strong subunit interactions. Future possibilities include verification of the proposed reaction mechanism and tracing of additional relationships, perhaps also with other protein families. Short-chain dehydrogenases illustrate the value of comparisons and diversified research in generating unexpected discoveries.
Amino Acid Sequence↗
Gaucher disease in Spanish patients: analysis of eight mutations.
Gaucher disease is particularly prevalent among Ashkenazi Jews; thus most studies have been reported on this ethnic group. We present the first data on Spanish patients with Gaucher disease and provide one of the first reports on a fairly well defined, large, non-Jewish population. Eight mutations were analyzed in 35 patients, with different clinical subtypes, by restriction enzyme digestion or allele-specific oligonucleotide (ASO) hybridization, after PCR amplification of genomic DNA. Analysis of the eight mutations allowed identification of 77.2% of the disease alleles, N370S and L444P alone accounting for 70%. Mutation N370S, carried by 31 alleles (44.3%), appeared to be the most prevalent in the Spanish population. The frequency of this mutation and of the N370S/N370S genotype is closer to those described for Ashkenazi Jews than to the frequencies found in other non-Jewish populations. Mutation L444P, the second most abundant mutation, occurred in 25.7% of the disease alleles. Four alleles carrying mutation D409H (5.7%) were detected in patients of different clinical expression and one RecNciI allele in a type I patient. Mutations 84GG, IVS2 + 1, R463C, and RecTL were also screened but were not found in any of our patients.
Adolescent↗
Evidence against involvement of recoverin in autosomal recessive retinitis pigmentosa in 42 Spanish families.
Autosomal recessive retinitis pigmentosa (ARRP) is a degenerative disease of photoreceptors in which defects in the genes encoding rhodopsin, the beta subunit of rod phosphodiesterase (PDEB) and, recently, in the gene for rod cGMP-gated channel, have been reported. However, detailed genetic involvement has not been ascertained in the great majority of cases. Recoverin, another member of the light transduction pathway, is a candidate gene for ARRP. We report the first analyses of the involvement of the recoverin gene (RCV1) in 42 Spanish ARRP families. Linkage and homozygosity studies with an intragenic polymorphism and the close markers D17S945 and D17S786 ruled out RCV1 as the cause of ARRP in 38 pedigrees. In the four remaining families, single strand conformation polymorphism analysis of the recoverin-coding region detected no mutations in the parents or in the affected members. These results strongly suggest that mutations in the RCV1 gene are not responsible for ARRP in these families.
Base Sequence↗
Unusual expression of Gaucher's disease: cardiovascular calcifications in three sibs homozygous for the D409H mutation.
Three sisters suffering from an unusual form of Gaucher's disease are described. These patients had cardiovascular abnormalities consisting of calcification of the ascending aorta and of the aortic and mitral valves. Neurological findings included ophthalmoplegia and saccadic eye movements in two patients, and tonic-clonic seizures in the third. The three patients died, two of them after having undergone aortic valve replacement. Tissue was obtained from one of the sibs and fibroblast and liver beta-glucocerebrosidase activity was reduced to 4% and 11% of mean normal values. Genotype analysis indicated that the patient was homozygous for the D409H mutation. It is tempting to relate the phenotype of severe cardiac involvement to the D409H/D409H genotype, although further cases will be needed before this association can be confirmed.
Adolescent↗
Structure of the Drosophila melanogaster glutathione-dependent formaldehyde dehydrogenase/octanol dehydrogenase gene (class III alcohol dehydrogenase). Evolutionary pathway of the alcohol dehydrogenase genes.
The glutathione-dependent formaldehyde dehydrogenase gene (gfd) of Drosophila melanogaster encodes an enzyme that is active toward S-hydroxymethylglutathione, an adduct of formaldehyde with glutathione, and also with long-chain primary alcohols, both properties typical of class III alcohol dehydrogenases, gfd hybridizes at the 86D division of the third chromosome, in agreement with the known location of the Drosophila octanol dehydrogenase gene (odh), gfd/odh was isolated from a lambda EMBL-4 genomic library and consists of three exons (with coding segments of 21, 90 and 1029 bp) and two introns (69 bp and 70 bp, respectively). The introns are small in size like the Drosophila interrupting sequences and are located at the 5' end of the coding region. Comparisons with the homologous genes of Saccharomyces, Candida and humans provide information on the evolution of the class III alcohol dehydrogenases. Moreover, results from analysis of exon/intron distributions in eleven dehydrogenases are compatible with the hypothesis of intron loss accounting for aspects of the present structure of these genes.
Alcohol Oxidoreductases↗
Fundamental molecular differences between alcohol dehydrogenase classes.
Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism.
Alcohol Dehydrogenase↗
Drosophila lebanonensis ADH: analysis of recombinant wild-type enzyme and site-directed mutants. The effect of restoring the consensus sequence in two positions.
Unique amino acid substitutions occur in D. lebanonensis ADH. They are found within the putative NAD(+)-binding domain and affect residues that are otherwise highly conserved in all other species of the genus. To restore the consensus amino acids, we have constructed an expression system for this enzyme in E. coli, and engineered two mutants, Ala13Gly and Asn56Thr. The biochemical and kinetic features of these retromutants are consistent with increased catalytic efficiency and thermal stability. Thus, results show that wild-type D. lebanonensis ADH can be improved by site-directed mutagenesis.
Alcohol Dehydrogenase↗
Genetic fine localization of the arrestin (S-antigen) gene 4 cM distal from D2S172.
Arrestin is a component of the light transduction cascade that takes place in the outer segment of retinal rods. In situ hybridization and linkage analysis have localized the arrestin gene to a region of 50 cM between CRYG alpha and D2S23/D2S55 on chromosome 2q24-37. We have performed pairwise and multipoint linkage analysis between arrestin and four highly polymorphic markers from this region. The results indicate tight linkage between the gene and the microsatellite D2S172 (Zmax = 9.25 at theta = 0.038). This fine localization of the gene should provide a useful tool for cosegregation analyses involving the arrestin gene.
Antigens↗
Analysis of nucleotide substitutions and amino acid conservation in the Drosophila Adh genomic region.
The homologous genomic region that contains two paralogous genes, Adh and Adh-dup, was compared in several Drosophila species. Sequences were analyzed as follows: a) At the nucleotide level, Ka and Ks values were determined for each pair of species. Ka-Adh and Ka-Adh-dup are not significantly different. However, Ks-Adh values are significantly lower than Ks-Adh-dup, which are more variable. In agreement with other reports, lower Ks values for Adh correlate with a high level of gene expression and relatively high percentage of G+C content in the third codon position, while the opposite applies to Adh-dup. b) At the protein level, amino acid comparisons reveal conserved regions shared by ADH and ADH-DUP, which have been assigned to known functional domains. Key residues for dehydrogenasic function are also found in ADH-DUP, thus pointing to a dehydrogenase activity for ADH-DUP, albeit very different from that of ADH.
Alcohol Dehydrogenase↗
Adh and Adh-dup sequences of Drosophila lebanonensis and D. immigrans: interspecies comparisons.
We have cloned and sequenced the Adh genomic region of Drosophila lebanonensis (subgenus Scaptodrosophila) and D. immigrans (subgenus Drosophila). This region, which contains Adh, encoding the alcohol dehydrogenase enzyme, and Adh-dup (duplicate of Adh), has been compared with the same fragment from D. subobscura (subgenus Sophophora). Even though the flanking regions and introns of both genes have been affected by high substitution rates, the consensus sequences have been clearly identified. Although the overall homology of the coding regions was 76-78% among the species compared, there were differences in the exon distribution of the nucleotide substitutions when Adh or Adh-dup were compared, thus showing that these two genes differ in their evolutionary pattern.
Alcohol Dehydrogenase↗
Preservation of antigenic reactivity after cryofixation, cryosubstitution, and cryoembedding in lowicryl HM23: application to alcohol dehydrogenase in Drosophila fat body.
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Acrylic Resins↗
Effect of site-directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase.
Tyr152 and Lys156 may be functionally important residues in Drosophila ADH as they are conserved in the genus and in all short-chain dehydrogenases. In addition, unaltered Gly positions could have a crucial role in the building of the structural framework. We have modified Drosophila ADH and expressed the mutant forms in E. coli. Mutation of Tyr152 to Glu or Gln, Lys156 to Ile, Gly184 to Leu, and the double mutant Gly130 to Cys and Gly133 to Ile, all rendered, with different substrates and at different pHs, an inactive enzyme. Results suggest that Tyr152 and Lys156 are involved in catalysis and that Gly130, Gly133 and Gly184 contribute substantially to the structure of the active form.
Alcohol Dehydrogenase↗
Evidence of serine-protease activity closely associated with Drosophila alcohol dehydrogenase.
With the use of monoclonal antibodies against alcohol dehydrogenase (ADH) we detected ADH proteolysis in different Drosophila melanogaster tissues during development [Visa, N., Fiblas, J., Santa-Crus, M. C. & Gonzàlez-Duarte R. (1992) J. Histochem. Cytochem. 40, 39-49]. We now report the analysis of this proteolytic activity in crude homogenates and in purified ADH preparations of several Drosophila species. Our results indicate that in non-denaturing IEF gels the proteolytic activity comigrates with native ADH electromorphs of all the species analyzed. In addition, we show that it copurifies with ADH and is responsible for the instability of apparently homogeneous ADH preparations in the presence of SDS. When purified ADH preparations were analyzed, the endogenous proteolytic activity yielded the same banding pattern as that obtained with crude homogenates. Even after rechromatography on Sephacryl S-200, the usual last step in our standard purification protocol, the proteolytic activity remained associated with the ADH fractions. Among the various agents which could explain the ADH-linked proteolytic effect, a pre-existing nicked state of the enzyme or chemical proteolysis have been ruled out. The kinetics observed on pure ADH preparations, the effect of specific protease inhibitors and substrate specificity have led us to ascribe this activity to the subtilase serine-protease family. Given that proteolysis is evident even in rechromatographed Sephacryl S-200 fractions, if incubated in SDS for enough time, we propose two alternative hypotheses to explain this phenomenon. First, the proteolytic activity may come from a protease which is inseparable from the ADH active forms and second, the ADH itself may behave as a subtilase when it adopts a particular conformation. Moreover, the previously reported differential banding pattern during development suggests a role for this activity in vivo, in which fatty acids could produce the inducer effect attributed to SDS in vitro.
Alcohol Dehydrogenase↗
Characterization and evolution of the Adh genomic region in Drosophila guanche and Drosophila madeirensis.
We have sequenced the genomic region of the Adh (alcohol dehydrogenase) gene of Drosophila guanche and Drosophila madeirensis. Two genes, Adh and Adh-dup, whose main features are shared by other Drosophila species, are contained in this region. Interspecies comparisons of this genomic region of these two species with the region in D. subobscura have been made. Our data are in agreement with the phylogenetic relationship of the three species and particularly with the very close proximity of D. madeirensis and D. subobscura. The correct alignment of the noncoding as well as the coding sequences allows us to infer how this region has evolved. The degree of divergence of D. madeirensis and D. subobscura Adh sequences is low and replacement substitutions are as frequent as silent substitutions. In addition, heterogeneous divergence rates for coding and noncoding sequences have been observed. Our data also reveal that a recent evolutionary event is responsible for the branching of the three species.
Alcohol Dehydrogenase↗
Colorimetric assay to determine alcohol dehydrogenase activity.
A colorimetric assay has been developed to quantify alcohol dehydrogenase (ADH) activity. The advantage of this method over conventional spectrophotometric assays is that many samples can be processed at once in a 96-well plate, monitoring the absorbance at 590 nm with a microtiter reader. ADH inhibitors have been used to show the specificity of this method. The assay offers an easy and reliable test for monitoring routine measurements of ADH activity and could be a valuable tool for those involved in the biomedical research of alcoholism, as well as in gene expression methodology in which Drosophila Adh has been widely used as reporter gene in transfection assays.
Alcohol Dehydrogenase↗