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1.
Microbiol Resour Announc ; 12(1): e0104922, 2023 Jan 24.
Article in English | MEDLINE | ID: mdl-36598284

ABSTRACT

We report the draft genome sequence of Bjerkandera adusta Dec 1, a basidiomycete that was isolated from the soil in Yokohama, Japan, using the Illumina HiSeq platform. B. adusta Dec 1 was identified as a fungus that degrades persistent anthraquinone dyes, and the novel peroxidase DyP was responsible for this degradation.

2.
Int J Mol Sci ; 22(16)2021 Aug 12.
Article in English | MEDLINE | ID: mdl-34445389

ABSTRACT

DyP-type peroxidases are a family of heme peroxidases named for their ability to degrade persistent anthraquinone dyes. DyP-type peroxidases are subclassified into three classes: classes P, I and V. Based on its genome sequence, Streptomyces avermitilis, eubacteria, has two genes presumed to encode class V DyP-type peroxidases and two class I genes. We have previously shown that ectopically expressed SaDyP2, a member of class V, indeed has the characteristics of a DyP-type peroxidase. In this study, we analyzed SaDyP1, a member of the same class V as SaDyP2. SaDyP1 showed high amino acid sequence identity to SaDyP2, retaining a conserved GXXDG motif and catalytic aspartate. SaDyP1 degraded anthraquinone dyes, which are specific substrates of DyP-type peroxidases but not azo dyes. In addition to such substrate specificity, SaDyP1 showed other features of DyP-type peroxidases, such as low optimal pH. Furthermore, immunoblotting using an anti-SaDyP2 polyclonal antibody revealed that SaDyP1 and/or SaDyP2 is expressed in mycelia of wild-type S. avermitilis.


Subject(s)
Peroxidases/genetics , Peroxidases/metabolism , Streptomyces/enzymology , Whole Genome Sequencing/methods , Amino Acid Motifs , Amino Acid Sequence , Anthraquinones/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enzyme Stability , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Genome, Bacterial , Hydrogen-Ion Concentration , Models, Molecular , Peroxidases/chemistry , Protein Conformation , Streptomyces/genetics , Thermodynamics
3.
AMB Express ; 9(1): 56, 2019 Apr 23.
Article in English | MEDLINE | ID: mdl-31016483

ABSTRACT

Alizarin is an anti-fungal compound produced by the plant, Rubia tinctorum. The parasitic fungus Bjerkandera adusta Dec 1 was cultured in potato dextrose (PD) medium with or without alizarin. Alizarin was a good substrate for the dye-decolorizing peroxidase (DyP) from B. adusta Dec 1 and hampered B. adusta growth at the early stage of plate culture. During liquid shaking culture, DyP activity in cultures supplemented with 100 µM alizarin was greater than that in controls cultured without alizarin. In particular, DyP activity per dry cell mass increased approximately 3.5-, 3.1-, and 2.9-fold at 24, 30, and 36 h after inoculation, respectively, compared with control cultures. These data suggest that alizarin stimulates the expression of DyP. Interestingly, alizarin rapidly decomposed at an early stage in culture (24-42 h) in PD medium supplemented with 100 µM alizarin. Thus, alizarin appears to induce DyP expression in B. adusta Dec 1, and this DyP, in turn, rapidly degrades alizarin. Collectively, our findings suggest that the physiological role of DyP is to degrade antifungal compounds produced by plants.

4.
J Biosci Bioeng ; 123(4): 425-430, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28089379

ABSTRACT

DyP-type peroxidases are a heme peroxidase family with unique properties whose members are widely distributed from prokaryotes to eukaryotes. DyP-type peroxidases are subdivided into class P, I and V based on structure-based sequence alignment. Class V enzymes possess degradation activities for anthraquinone dyes, and include extra sequences compared with class P and I. Class V enzymes are mainly found in fungi, with only two such proteins, AnaPX and DyP2, reported in bacteria. Here, we heterologously expressed, purified and biochemically characterized SaDyP2 protein, predicted to belong to class V. SaDyP2 was purified as a ∼50 kDa enzyme containing a heme cofactor and was found to oxidize the typical peroxidase substrates, ABTS and DMP. SaDyP2 was generally thermostable and exhibited a lower optimal pH, a feature typical of DyP-type peroxidases. It also degraded anthraquinone dyes, a specific substrate of DyP-type peroxidases, although the kcat for SaDyP2 was lower than that for other class V enzymes. The Km value of SaDyP2 for anthraquinone dye was similar to that of other enzymes of this class. Homology modeling revealed that the structure of SaDyP2 best fit that of class V enzymes.


Subject(s)
Peroxidase/classification , Peroxidase/metabolism , Streptomyces/enzymology , Amino Acid Sequence , Anthraquinones/metabolism , Coloring Agents/metabolism , Enzyme Stability , Heme/metabolism , Hydrogen-Ion Concentration , Kinetics , Oxidation-Reduction , Peroxidase/chemistry , Peroxidase/isolation & purification , Sequence Alignment , Substrate Specificity
5.
FEBS Lett ; 589(19 Pt B): 2707-12, 2015 Sep 14.
Article in English | MEDLINE | ID: mdl-26297831

ABSTRACT

Mitochondrial ATP synthase is a motor enzyme in which a central shaft rotates in the stator casings fixed with the peripheral stator stalk. When expression of d-subunit, a stator stalk component, was knocked-down, human cells could not form ATP synthase holocomplex and instead accumulated two subcomplexes, one containing a central rotor shaft plus catalytic subunits (F1-c-ring) and the other containing stator stalk components ("b-e-g" complex). F1-c-ring was also formed when expression of mitochondrial DNA-coded a-subunit and A6L was suppressed. Thus, the central rotor shaft and the stator stalk are formed separately and they assemble later. Similar assembly strategy has been known for ATP synthase of yeast and Escherichia coli and could be common to all organisms.


Subject(s)
Mitochondrial Proton-Translocating ATPases/chemistry , Protein Aggregates , Gene Knockdown Techniques , HeLa Cells , Humans , Mitochondrial Proteins/biosynthesis , Mitochondrial Proton-Translocating ATPases/genetics , Mitochondrial Proton-Translocating ATPases/metabolism , Protein Subunits/deficiency , Protein Subunits/genetics
6.
Genes Cells ; 19(2): 153-60, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24330338

ABSTRACT

A 6.8-kDa proteolipid (called MLQ) is a hydrophobic mitochondrial protein with unknown function that is loosely associated with ATP synthase. Here, we show that MLQ-knockdown HeLa cells lose population of ATP synthase in mitochondria. This is not due to low transcription of subunit genes of ATP synthase because levels of mRNA for α- and ß-subunits are unaffected by the knockdown. As a consequence, the knockdown cells show low mitochondrial ATP synthesis activity, grow slowly in the normal medium, and are vulnerable to glucose deprivation. Given that the expression of MLQ varies responding to cellular conditions, MLQ is a potential regulator of the mitochondrial ATP synthesis.


Subject(s)
Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Proteolipids/metabolism , Proton-Translocating ATPases/metabolism , Cell Enlargement , Cell Survival , Electron Transport Chain Complex Proteins/metabolism , HeLa Cells , Humans , Membrane Potential, Mitochondrial , Mitochondria/genetics , Mitochondrial Proteins/genetics , Mitochondrial Proton-Translocating ATPases , Proteolipids/genetics , Proton-Translocating ATPases/genetics , RNA, Messenger/metabolism
7.
FEBS Lett ; 587(23): 3843-7, 2013 Nov 29.
Article in English | MEDLINE | ID: mdl-24157360

ABSTRACT

Mitochondrial ATP synthase, a major ATP supplier in respiring cells, should be regulated in amount and in activity to respond to the varying demands of cells for ATP. We screened 80 protein kinase inhibitors and found that HeLa cells treated with four inhibitors exhibited reduced mitochondrial ATP synthesis activity. Consistently, knockdown of their target kinases (PKA, PKCδ, CaMKII and smMLCK) resulted in a decrease in mitochondrial ATP synthesis activity. Among them, mitochondria of smMLCK-knockdown cells contained only a small amount of ATP synthase, while the α- and ß-subunits of ATP synthase were produced normally, suggesting that smMLCK affects assembly (or decay) of ATP synthase.


Subject(s)
Adenosine Triphosphate/biosynthesis , Mitochondria/metabolism , Myosin-Light-Chain Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , HeLa Cells , Humans , Mitochondria/drug effects , Mitochondrial Proton-Translocating ATPases/genetics , Mitochondrial Proton-Translocating ATPases/metabolism , Myosin-Light-Chain Kinase/genetics , Myosin-Light-Chain Kinase/metabolism , Protein Kinase C-delta/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Small Interfering
8.
Mol Genet Metab ; 100(3): 257-61, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20409739

ABSTRACT

Fabry disease is an X-linked genetic disorder caused by a deficiency of alpha-galactosidase A (GLA) activity. As enzyme replacement therapy (ERT) involving recombinant GLAs has been introduced for this disease, a useful biomarker for diagnosis and monitoring of therapy has been strongly required. We measured globotriaosylsphingosine (lyso-Gb3) and globotriaosylceramide (Gb3) in plasma samples from ten hemizygous males (six classic and four variant cases) and eight heterozygous females with Fabry disease, and investigated the responses of plasma lyso-Gb3 and Gb3 in a male Fabry patient who had undergone ERT for 4years to determine whether plasma lyso-Gb3 and Gb3 could be biomarkers of Fabry disease. The results revealed that plasma lyso-Gb3 was apparently increased in male patients and was higher in cases of the classic form than those of the variant one. In Fabry females, plasma lyso-Gb3 was moderately increased in both symptomatic and asymptomatic cases, and there was a correlation between the increase in lyso-Gb3 and the decrease in GLA activity. As to plasma Gb3, the levels in the variant Fabry hemizygotes and Fabry heterozygotes could not be distinguished from those in the controls, although those in the classic Fabry hemizygotes were increased. The plasma lyso-Gb3 level in the Fabry patient who had received ERT was elevated at the baseline and fell more dramatically on ERT than that of Gb3. Plasma lyso-Gb3 could thus be a potential biomarker of Fabry disease.


Subject(s)
Fabry Disease/blood , Glycolipids/blood , Sphingolipids/blood , Trihexosylceramides/blood , Adolescent , Adult , Aged , Biomarkers/blood , Case-Control Studies , Fabry Disease/drug therapy , Fabry Disease/genetics , Female , Heterozygote , Humans , Male , Middle Aged , Recombinant Proteins/therapeutic use , Young Adult , alpha-Galactosidase/blood , alpha-Galactosidase/genetics , alpha-Galactosidase/therapeutic use
9.
J Hum Genet ; 55(3): 175-8, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20139917

ABSTRACT

Fabry disease is a genetic disorder caused by a deficiency of alpha-galactosidase, exhibiting a wide clinical spectrum, from the early-onset severe 'classic' form to the late-onset mild 'variant' one. Recent screening of newborns revealed that the incidence of Fabry disease is unexpectedly high, and that the genotypes of patients with this disease are quite heterogeneous and many novel mutations have been identified in them. This suggests that a lot of Fabry patients will be found in an early clinical stage when the prognosis is obscure and a proper therapeutic schedule for them cannot be determined. Thus, it is significant to predict the clinical phenotype of this disease resulting from a novel mutation. Herein, we proposed a phenotype prediction model based on sequential and structural information. As far as we know, this is the first report of phenotype prediction for Fabry disease. First, we investigated the sequential and structural changes in the alpha-galactosidase molecule responsible for Fabry disease. The results showed that there are quite large differences in several properties between the classic and variant groups. We then developed a phenotype prediction model involving the decision tree technique. The accuracy of this prediction model is high (86%), and Matthew's correlation coefficient is also high (0.49). The phenotype predictor proposed in this paper may be useful for determining a proper therapeutic schedule for this disease.


Subject(s)
Fabry Disease/diagnosis , Fabry Disease/enzymology , Sequence Analysis, Protein , alpha-Galactosidase/chemistry , Databases, Genetic , Decision Trees , Fabry Disease/pathology , Humans , Models, Genetic , Mutation/genetics , Phenotype
10.
Brain Dev ; 32(7): 524-30, 2010 Aug.
Article in English | MEDLINE | ID: mdl-19793631

ABSTRACT

To elucidate the basis of neuronal ceroid lipofuscinosis 1 (CLN1) from the viewpoint of enzyme structure, we constructed structural models of mutant palmitoyl protein thioesterase 1 (PPT1) proteins using molecular modeling software, jackal and TINKER. We classified the amino acid substitutions responsible for CLN1 and divided them into two groups, groups 1 and 2, based on the biochemical phenotype. Then, we examined the structural changes in the PPT1 protein for each group by calculating the solvent-accessible surface area (ASA) and the number of atoms affected. Our results revealed that the structural changes in group 1, which exhibits a complete deficiency of PPT1 activity, were generally large and located in the core region of the enzyme molecule. In group 2 exhibiting residual PPT1 activity, the structural changes in PPT1 were smaller and localized near the surface of the enzyme molecule. Coloring of affected atoms based on the distances between those in the wild type and mutants revealed the characteristic structural changes in the PPT1 protein geographically and semi-quantitatively. Structural investigation provides us with a deeper insight into the basis of CLN1.


Subject(s)
Membrane Proteins/chemistry , Models, Molecular , Neuronal Ceroid-Lipofuscinoses/enzymology , Amino Acid Substitution , Animals , Cattle , Humans , Membrane Proteins/genetics , Software , Thiolester Hydrolases
11.
Am J Hum Genet ; 85(5): 569-80, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19853240

ABSTRACT

A modified alpha-N-acetylgalactosaminidase (NAGA) with alpha-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-alpha-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease.


Subject(s)
Enzyme Replacement Therapy/methods , Fabry Disease/drug therapy , alpha-N-Acetylgalactosaminidase/chemistry , alpha-N-Acetylgalactosaminidase/therapeutic use , Amino Acid Substitution , Animals , Binding Sites , CHO Cells , Catalysis , Cells, Cultured , Cricetinae , Cricetulus , Culture Media, Conditioned/chemistry , DNA, Complementary/metabolism , Disease Models, Animal , Drug Stability , Fabry Disease/enzymology , Fabry Disease/metabolism , Fibroblasts/drug effects , Fluorescent Dyes/metabolism , Galactosides/metabolism , Genetic Vectors , Humans , Hydrogen-Ion Concentration , Hymecromone/analogs & derivatives , Hymecromone/metabolism , Immunohistochemistry , Kidney/drug effects , Kidney/pathology , Kidney/ultrastructure , Liver/drug effects , Liver/pathology , Liver/ultrastructure , Mice , Mice, Knockout , Models, Molecular , Molecular Weight , Myocardium/pathology , Myocardium/ultrastructure , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/therapeutic use , Retroviridae/genetics , Transfection , Trihexosylceramides/metabolism , alpha-N-Acetylgalactosaminidase/genetics , alpha-N-Acetylgalactosaminidase/isolation & purification
12.
J Hum Genet ; 54(9): 510-5, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19644515

ABSTRACT

Allelic mutations of the lysosomal beta-galactosidase gene cause heterogeneous clinical phenotypes, such as GM1 gangliosidosis and Morquio B disease, the former being further classified into three variants, namely infantile, juvenile and adult forms; and heterogeneous biochemical phenotypes were shown in these forms. We tried to elucidate the bases of these diseases from a structural viewpoint. We first constructed a three-dimensional structural model of human beta-galactosidase by means of homology modeling. The human beta-galactosidase consists of three domains, such as, a TIM barrel fold domain, which functions as a catalytic domain, and two galactose-binding domain-like fold domains. We then constructed structural models of representative mutant beta-galactosidase proteins (G123R, R201C, I51T and Y83H) and predicted the structural change associated with each phenotype by calculating the number of affected atoms, determining the root-mean-square deviation and the solvent-accessible surface area, and by color imaging. The results show that there is a good correlation between the structural changes caused by amino-acid substitutions in the beta-galactosidase molecule, as well as biochemical and clinical phenotypes in these representative cases. Protein structural study is useful for elucidating the bases of these diseases.


Subject(s)
Gangliosidosis, GM1/genetics , Mucopolysaccharidosis IV/genetics , Mutation/genetics , beta-Galactosidase/chemistry , beta-Galactosidase/genetics , Amino Acid Sequence , Amino Acid Substitution , Humans , Models, Molecular , Molecular Sequence Data , Phenotype , Protein Conformation , Sequence Homology, Amino Acid
13.
J Hum Genet ; 54(6): 324-30, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19343043

ABSTRACT

To elucidate the mechanism underlying transport and processing defects from the viewpoint of enzyme folding, we constructed three-dimensional models of human acid alpha-glucosidase encompassing 27 relevant amino acid substitutions by means of homology modeling. Then, we determined in each separate case the number of affected atoms, the root-mean-square distance value and the solvent-accessible surface area value. The analysis revealed that the amino acid substitutions causing a processing or transport defect responsible for Pompe disease were widely spread over all of the five domains comprising the acid alpha-glucosidase. They were distributed from the core to the surface of the enzyme molecule, and the predicted structural changes varied from large to very small. Among the structural changes, we paid particular attention to G377R and G483R. These two substitutions are predicted to cause electrostatic changes in neighboring small regions on the molecular surface. The quality control system of the endoplasmic reticulum apparently detects these very small structural changes and degrades the mutant enzyme precursor (G377R), but also the cellular sorting system might be misled by these minor changes whereby the precursor is secreted instead of being transported to lysosomes (G483R).


Subject(s)
Glycogen Storage Disease Type II/enzymology , Mutant Proteins/chemistry , Protein Processing, Post-Translational , Protein Transport , alpha-Glucosidases/chemistry , Amino Acid Sequence , Amino Acid Substitution , Endoplasmic Reticulum/metabolism , Glycogen Storage Disease Type II/genetics , Humans , Lysosomes/metabolism , Models, Molecular , Molecular Sequence Data , Mutant Proteins/metabolism , Protein Conformation , Sequence Homology, Amino Acid , alpha-Glucosidases/metabolism
14.
Mol Genet Metab ; 96(4): 233-8, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19181556

ABSTRACT

Enzyme enhancement therapy (EET) for Fabry disease involving imino sugars has been developed and attracted interest. It is thought that imino sugars act as pharmacological chaperones for wild-type and mutant alpha-galactosidases (GLAs) in cells, but the mechanisms underlying the molecular interactions between the imino sugars and the enzyme have not been clarified yet. We examined various kinds of imino sugars and found that galactostatin bisulfite (GBS) inhibited GLA in vitro and increased the enzyme activity in cultured Fabry fibroblasts as in the case of 1-deoxygalactonojirimycin (DGJ). Then, we analyzed the molecular interactions between the imino sugars and recombinant human GLA by means of isothermal titration calorimetry and surface plasmon resonance biosensor assays, and first determined the thermodynamic and binding-kinetics parameters of imino sugar and GLA complex formation. The results revealed that DGJ bound to the enzyme more strongly than GBS, the binding of DGJ to the enzyme protein being enthalpy-driven. In the case of GBS, the reaction was mainly enthalpy-driven, but there was a possibility that entropy-driven factors were involved in the binding. Structural analysis in silico revealed that both the chemicals fit into the active-site pocket and undergo hydrogen bonding with residues comprising the active-site pocket including the catalytic ones. The side chain of GBS was oriented towards the entrance of the active-site pocket, and thus it could be in contact with residues comprising the wall of the active-site pocket. Thermodynamic, kinetic and structural studies should provide us with a lot of information for improving EET for Fabry disease.


Subject(s)
Fabry Disease/enzymology , Imino Sugars/pharmacology , alpha-Galactosidase/antagonists & inhibitors , 1-Deoxynojirimycin/analogs & derivatives , 1-Deoxynojirimycin/chemistry , 1-Deoxynojirimycin/pharmacology , Animals , CHO Cells , Catalytic Domain , Cells, Cultured , Cricetinae , Cricetulus , Fabry Disease/pathology , Fibroblasts/drug effects , Fibroblasts/enzymology , Fibroblasts/pathology , Galactosamine/analogs & derivatives , Galactosamine/chemistry , Galactosamine/pharmacology , Humans , Imino Sugars/chemistry , Kinetics , Models, Molecular , Thermodynamics , alpha-Galactosidase/metabolism
15.
Biochem Biophys Res Commun ; 377(4): 1168-72, 2008 Dec 26.
Article in English | MEDLINE | ID: mdl-18992224

ABSTRACT

To elucidate the basis of aspartylglucosaminuria (AGU) from the viewpoint of enzyme structure, we constructed structural models of mutant aspartylglucosaminidase (AGA) proteins using molecular modeling software, TINKER. We classified the amino acid substitutions responsible for AGU and divided them into three groups based on the biochemical phenotype. Then, we examined the structural changes in the AGA protein for each group by calculating the solvent-accessible surface area (ASA), the number of atoms affected, and the root-mean-square deviation (RMSD). Our results revealed that the structural changes in group 1, which exhibits folding/transport defects and a complete deficiency of AGA activity, were generally large and located in the core region of the enzyme molecule. In group 2, exhibiting the mature AGA protein but no AGA activity, the functionally important region of the enzyme molecule was seriously affected. In group 3 exhibiting residual AGA activity, the structural changes in AGA were small and localized near the surface of the enzyme molecule. Coloring of affected atoms based on the distances between the wild-type and mutant ones revealed the characteristic structural changes in the AGA protein geographically and semi-quantitatively. Structural investigation provides us with a deeper insight into the basis of AGU.


Subject(s)
Aspartylglucosaminuria/genetics , Aspartylglucosylaminase/chemistry , Computer Simulation , Models, Molecular , Amino Acid Sequence , Amino Acid Substitution , Aspartylglucosaminuria/enzymology , Aspartylglucosylaminase/classification , Aspartylglucosylaminase/genetics , Humans , Molecular Sequence Data , Mutation , Protein Structure, Secondary , Software
16.
Biol Pharm Bull ; 31(9): 1691-5, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18758061

ABSTRACT

To examine the uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts from a patient with mucopolysaccharidosis I (MPS I) and its effect on the cleavage of accumulated substrates, we performed enzymological, Western blotting, immunocytochemical and morphological studies. Laronidase was incorporated into the MPS I cells dose-dependently mainly via mannose 6-phosphate (M6P) receptors. Then the incorporated enzyme was transported to lysosomes and processed to the mature form, the pathological changes of the cells being improved. Furthermore, we compared the uptake of laronidase by cultured mouse osteoblasts with that by cultured mouse fibroblasts. The enzyme was incorporated into the cultured mouse osteoblasts mainly via M6P receptors, although mannose (Man) receptors were partially involved in the uptake of the enzyme, as in the cultured fibroblasts. But the uptake by the former was apparently lower than that by the latter. The administration of a high dose of the enzyme or development of a recombinant alpha-L-iduronidase containing many M6P residues is required for further improvement of enzyme replacement therapy for skeletal disorders caused by MPS I.


Subject(s)
Fibroblasts/metabolism , Iduronidase/metabolism , Osteoblasts/metabolism , Animals , Cells, Cultured , Fibroblasts/ultrastructure , Humans , Kinetics , Mice , Mucopolysaccharidosis I/metabolism , Osteoblasts/ultrastructure , Receptor, IGF Type 2/metabolism , Recombinant Proteins/metabolism
17.
J Hum Genet ; 53(9): 812-824, 2008.
Article in English | MEDLINE | ID: mdl-18633574

ABSTRACT

Fabry disease is an inborn error of glycolipid catabolism resulting from lesions in the gene encoding alpha-galactosidase (GLA). To elucidate the basis of Fabry disease, we constructed structural models of mutant GLAs responsible for the disease and calculated indexes, i.e., the numbers of atoms affected in the main chain and side chain of each mutant GLA, the root-mean-square distance values, and the solvent-accessible surface-area values, based on 212 Fabry amino acid substitutions previously reported (196 classic and 16 variant). As two therapeutic options, enzyme replacement and enzyme enhancement, are now available for this disease, proper prediction of the natural outcome and therapeutic efficiency based on the molecular evidence for individual cases are critical for patients' quality of life. Our results revealed that structural changes in the classic Fabry group were generally large and tended to be in the core region of a protein or located in the functionally important region, including the active-site pocket. On the other hand, structural changes in the variant Fabry group were small or localized on the surface of the molecule far away from the active site. We focused on structural changes due to amino acid substitutions for which substrate analogues are effective for improving the stability or transportation of mutant GLAs, and the results of the study revealed that they are small or localized on the molecular surface, regardless of the phenotype. Coloring of affected atoms based on distances between wild type and mutant ones clearly showed the characteristic structural changes in the GLA protein geographically and subquantitatively. Structural investigation is useful for elucidation of the basis of Fabry disease and predicting disease outcome.


Subject(s)
Fabry Disease/genetics , alpha-Galactosidase/chemistry , alpha-Galactosidase/genetics , Amino Acid Substitution/genetics , Fabry Disease/enzymology , Humans , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/physiology , Mutation, Missense , Protein Conformation
18.
Mol Genet Metab ; 94(4): 462-468, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18490185

ABSTRACT

To determine the structural changes in the alpha-subunit of beta-hexosaminidase due to amino acid substitutions causing Tay-Sachs disease, we built structural models of mutant alpha-subunits resulting from 33 missense mutations (24 infantile and 9 late-onset), and analyzed the influence of each amino acid replacement on the structure by calculating the number of atoms affected and determining the solvent-accessible surface area of the corresponding amino acid residue in the wild-type alpha-subunit. In the infantile Tay-Sachs group, the number of atoms influenced by a mutation was generally larger than that in the late-onset Tay-Sachs group in both the main chain and the side chain, and residues associated with the mutations found in the infantile Tay-Sachs group tended to be less solvent-accessible than those in the late-onset Tay-Sachs group. Furthermore, color imaging determined the distribution and degree of the structural changes caused by representative amino acid substitutions, and that there were also differences between the infantile and late-onset Tay-Sachs disease groups. Structural study is useful for elucidating the basis of Tay-Sachs disease.


Subject(s)
Amino Acid Substitution , Hexosaminidase A/chemistry , Tay-Sachs Disease/enzymology , Tay-Sachs Disease/genetics , Adolescent , Adult , Age of Onset , Amino Acid Sequence , Child , Child, Preschool , Female , Hexosaminidase A/genetics , Hexosaminidase A/metabolism , Humans , Infant , Male , Models, Molecular , Mutation, Missense , Phenotype , Protein Conformation
19.
Clin Chim Acta ; 391(1-2): 68-73, 2008 May.
Article in English | MEDLINE | ID: mdl-18328816

ABSTRACT

BACKGROUND: Recently, enzyme enhancement therapy (EET) for Pompe disease involving imino sugars, which act as potential inhibitors of acid alpha-glucosidases in vitro, to improve the stability and/or transportation of mutant acid alpha-glucosidases in cells was studied and attracted interest. However, the mechanism underlying the molecular interaction between the imino sugars and the enzyme has not been clarified yet. METHODS: We examined the inhibitory and binding effects of four imino sugars on a recombinant human acid alpha-glucosidase, alglucosidase alfa, by means of inhibition assaying and isothermal titration calorimetry (ITC). Furthermore, we built structural models of complexes of the catalytic domain of the enzyme with the imino sugars bound to its active site by homology modeling, and examined the molecular interaction between them. RESULTS: All of the imino sugars examined exhibited a competitive inhibitory action against the enzyme, 1-deoxynojirimycin (DNJ) exhibiting the strongest action among them. ITC revealed that one compound molecule binds to one enzyme molecule and that DNJ most strongly binds to the enzyme among them. Structural analysis revealed that the active site of the enzyme is almost completely occupied by DNJ. CONCLUSION: These biochemical and structural analyses increased our understanding of the molecular interaction between a human acid alpha-glucosidase and imino sugars.


Subject(s)
Glycogen Storage Disease Type II/enzymology , Imino Sugars/metabolism , alpha-Glucosidases/metabolism , 1-Deoxynojirimycin/chemistry , 1-Deoxynojirimycin/metabolism , 1-Deoxynojirimycin/pharmacology , Binding Sites , Catalytic Domain , Drug Interactions , Glycoside Hydrolase Inhibitors , Humans , Imino Sugars/chemistry , Imino Sugars/pharmacology , Models, Molecular , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Thermodynamics
20.
J Hum Genet ; 53(5): 467-474, 2008.
Article in English | MEDLINE | ID: mdl-18340403

ABSTRACT

To elucidate the basis of mucopolysaccharidosis type I (MPS I), we constructed structural models of mutant alpha-L: -iduronidases (IDUAs) resulting from 33 amino acid substitutions that lead to MPS I (17 severe, eight intermediate, and eight attenuated). Then, we examined the structural changes in the enzyme protein by calculating the number of atoms affected and determined the root-mean-square distance (RMSD) and the solvent-accessible surface area (ASA). In the severe MPS I group, the number of atoms influenced by a mutation and the average RMSD value were larger than those in the attenuated group, and the residues associated with the mutations identified in the severe group tended to be less solvent accessible than those in the attenuated group. The clinically intermediate phenotype group exhibited intermediate values for the numbers of atoms affected, RMSD, and ASA between those in the severe group and those in the attenuated group. The results indicated that large structural changes had occurred in the core region in the severe MPS I group and small ones on the molecular surface in the attenuated MPS I group. Color imaging revealed the distributions and degrees of the structural changes caused by representative mutations for MPS I. Thus, structural analysis is useful for elucidating the basis of MPS I. As there was a difference in IDUA structural change between the severe MPS I group and the attenuated one, except for a couple of mutations, structural analysis can help predict the clinical outcome of the disease.


Subject(s)
Amino Acid Substitution/genetics , Iduronidase/chemistry , Iduronidase/genetics , Mucopolysaccharidosis I/genetics , Mutation, Missense , Humans , Iduronidase/physiology , Models, Molecular , Mucopolysaccharidosis I/enzymology
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