The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Hitoshi Sakuraba - One of the best experts on this subject based on the ideXlab platform.
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Elimination of abnormal sialylglycoproteins in fibroblasts with sialidosis and Galactosialidosis by normal gene transfer and enzyme replacement
Glycobiology, 2005Co-Authors: Yukako Oheda, Mai Murata, Yoshito Kadota, Masaharu Kotani, Yutaka Tatano, Jun Kuwahara, Hitoshi Sakuraba, Kohji Itoh, Yoshito Kadota, Kohji ItohAbstract:Sialidosis and Galactosialidosis are lysosomal storage diseases caused by the genetic defects of lysosomal sialidase (neuraminidase-1; NEU1) and lysosomal protective protein/cathepsin A (PPCA), respectively, associated with a NEU1 deficiency, excessive accumulation of sialylglycoconjugates, and development of progressive neurosomatic manifestations; in addition, the latter disorder is accompanied by simultaneous deficiencies of {szligbeta}-galactosidase and cathepsin A. We demonstrated that a few soluble N-glycosylated proteins carrying sialyloligosaccharides sensitive to glycopeptidase F (GPF) can be specifically detected in cultured fibroblasts from sialidosis and Galactosialidosis cases by blotting with a Maackia amurensis (MAM) lectin. We also examined the therapeutic effects of normal gene transfer and enzyme replacement by evaluating the decreases in sialylglycoconjugates accumulated in fibroblasts with these NEU1 deficiencies. The specific N-glycosylated proteins detected on MAM lectin blotting as well as the granular lysosomal fluorescence due to an avidin-FITC/biotinylated MAM lectin conjugate in sialidosis and Galactosialidosis fibroblasts disappeared in parallel with the restoration of the intracellular NEU1 activity after transfection of the recombinant NEU1 fused to HA tag sequence and the wild-type PPCA cDNA as well as administration of the recombinant PPCA precursor protein. The detection method for the abnormal sialylglycoproteins in cultured cells involving MAM lectin was demonstrated to be useful not only for biochemical and diagnostic analyses of NEU1 deficiencies but also for therapeutic evaluation of these conditions.
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cytochemical and biochemical detection of intracellularly accumulated sialyl glycoconjugates in sialidosis and Galactosialidosis fibroblasts with macckia amurensis
Clinica Chimica Acta, 2004Co-Authors: Masaharu Kotani, Hideo Yamada, Hitoshi SakurabaAbstract:Background: To clarify the pathogenesis of and evaluate experimental therapeutic trials for lysosomal diseases, effective tools for the detection of intracellularly accumulated materials are required. Methods: We examined a series of lectins for staining and blotting of the accumulated glycoconjugates in sialidosis and Galactosialidosis. Results: Lysosomally accumulated sialyl glycoconjugates were successfully detected in cultured fibroblasts from patients with these diseases by means of staining and blotting with Macckia amurensis (MAM). Conclusions: This procedure is sensitive and easy, and will be useful not only for biochemical and diagnostic analyses, but also for therapeutic evaluation in these diseases.
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endothelin 1 in the brain of patients with Galactosialidosis its abnormal increase and distribution pattern
Annals of Neurology, 2000Co-Authors: Kohji Itoh, Kiyomitsu Oyanagi, Michie Shimmoto, Hitoshi Takahashi, Takeshi Sato, Yoshio Hashizume, Hitoshi SakurabaAbstract:Endothelin-1 is a peptidic substrate in vitro of lysosomal protective protein/cathepsin A (PPCA) with serine carboxypeptidase activity. Endothelin-1-specific immunoreactivity has been demonstrated to be markedly increased and distributed abnormally in the neurons and glial cells within autopsied brain regions, including the cerebellum, hippocampal formation, and spinal cord, of patients affected with Galactosialidosis, a human PPCA deficiency. The genetic defect of the endothelin-1 degrading activity of PPCA is suggested to cause some of the neurological abnormalities of this disease.
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Expression of protective protein in human tissue.
Pediatric Neurology, 1999Co-Authors: Osamu Sohma, Akira Satake, Masashi Mizuguchi, Hitoshi Sakuraba, Kohji Itoh, Yoshiyuki Suzuki, Sachio Takashima, Kohji Itoh, Kiyomitsu OyanagiAbstract:The authors investigated by immunohistochemistry the distribution of protective protein in human tissues. Immunoreactivity was observed in the cytoplasm, revealing a granular pattern and cell type specificity. The most intense staining was observed in the large neurons of brain, distal and collecting tubular cells of kidney, epithelial cells of bronchus, and Leydig cells of testis. In a patient with Galactosialidosis type IIa, all these stains were absent. The neurons that were most strongly stained in the control group, such as the Betz cells, neurons in the basal forebrain, motor neurons in the cranial nerve nuclei, and ventral horn cells of the spinal cord, were markedly ballooned in the patient with Galactosialidosis.
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protective protein cathepsin a loss in cultured cells derived from an early infantile form of Galactosialidosis patients homozygous for the a1184 g transition y395c mutation
Biochemical and Biophysical Research Communications, 1998Co-Authors: Kohji Itoh, Norio Miharu, Koso Ohama, Nobuyuki Mizoguchi, Michie Shimmoto, Kouichi Utsumi, Hitoshi SakurabaAbstract:Abstract Galactosialidosis is a human autosomal recessive lysosomal storage disease caused by a genetic defect of protective protein/cathepsin A (PPCA). The patients in a Japanese family with the severe early-infantile form of Galactosialidosis were revealed to be homozygous for the A1184-G transition in the PPCA gene in both alleles, which leads to the Y395C substitution. The acid carboxypeptidase (cathepsin A) and lysosomal neuraminidase activities were markedly decreased in cultured fibroblasts and chorionic villus cells derived from the patients, although the decrease in β-galactosidase activity was less. Immunoblot and immunocytochemical analyses showed that neither the precursor nor the mature form of the PPCA gene product was present in the cultured cells. The Y395C mutation was revealed to cause the loss of the translated product, that determines the severity of the clinical phenotype.
Kohji Itoh - One of the best experts on this subject based on the ideXlab platform.
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a new heterozygous compound mutation in the ctsa gene in Galactosialidosis
Human genome variation, 2019Co-Authors: Kohji Itoh, Eiji Nanba, Hideki Nakajima, Miki Ueno, Kaori Adachi, Aya Narita, Jun Tsukimoto, A KawakamiAbstract:Galactosialidosis is an autosomal recessive lysosomal storage disease caused by the combined deficiency of lysosomal β-galactosidase and neuraminidase due to a defect in the protective protein/cathepsin A. Patients present with various clinical manifestations and are classified into three types according to the age of onset: the early infantile type, the late infantile type, and the juvenile/adult type. We report a Japanese female case of juvenile/adult type Galactosialidosis. Clinically, she presented with short stature, coarse facies, angiokeratoma, remarkable action myoclonus, and cerebellar ataxia. The patient was diagnosed with Galactosialidosis with confirmation of impaired β-galactosidase and neuraminidase function in cultured skin fibroblasts. Sanger sequencing for CTSA identified a compound heterozygous mutation consisting of NM_00308.3(CTSA):c.746 + 3A>G and c.655-1G>A. Additional analysis of her mother’s DNA sequence indicated that the former mutation originated from her mother, and therefore the latter was estimated to be from the father or was a de novo mutation. Both mutations are considered pathogenic owing to possible splicing abnormalities. One of them (c.655-1G>A) is novel because it has never been reported previously.
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use of modified u1 small nuclear rna for rescue from exon 7 skipping caused by 5 splice site mutation of human cathepsin a gene
Gene, 2018Co-Authors: Naoshi Yamazaki, Kohji Itoh, Keisuke Kanazawa, Maria Kimura, Makiko Shinomiya, Shouko Tanaka, Yasuo Shinohara, Noriaki Minakawa, Yoshiharu TakiguchiAbstract:Cathepsin A (CTSA) is a multifunctional lysosomal enzyme, and its hereditary defect causes an autosomal recessive disorder called Galactosialidosis. In a certain number of Galactosialidosis patients, a base substitution from adenine to guanine is observed at the +3 position of the 7th intron (IVS7 +3a>g) of the CTSA gene. With this mutation, a splicing error occurs; and consequently mRNA lacking the 7th exon is produced. This skipping of exon 7 causes a frame shift of the transcripts, resulting in a non-functional CTSA protein and hence Galactosialidosis. This mutation seems to make the interaction between the 5’-splice site of intron 7 of pre-mRNA and U1 small nuclear RNA (U1 snRNA) much weaker. In the present study, to produce properly spliced mRNA from the CTSA gene harboring this IVS7 +3a>g mutation, we examined the possible usefulness of modified U1 snRNA that could interact with the mutated 5’-splice site. Toward this goal, we first prepared a model system using a mutant CTSA mini gene plasmid for delivery into HeLa cells. Then, we examined the effectiveness of modified U1 snRNA on the formation of properly spliced mRNA from this mutant CTSA mini gene. As a result, we succeeded in obtaining improved formation of properly spliced CTSA mRNA. Our results suggest the usefulness of modified U1 snRNA for rescue from exon 7 skipping caused by the IVS7 +3a>g mutation of the CTSA gene.
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enzymatic activity of lysosomal carboxypeptidase cathepsin a is required for proper elastic fiber formation and inactivation of endothelin 1
Circulation, 2008Co-Authors: Volkan Seyrantepe, Carlos R. Morales, Kohji Itoh, Yoshito Kadota, Aleksander Hinek, Junzheng Peng, Michael Fedjaev, Sheila Ernest, Maryssa Canuel, Julie L. LavoieAbstract:BACKGROUND: Lysosomal carboxypeptidase, cathepsin A (protective protein, CathA), is a component of the lysosomal multienzyme complex along with beta-galactosidase (GAL) and sialidase Neu1, where it activates Neu1 and protects GAL and Neu1 against the rapid proteolytic degradation. On the cell surface, CathA, Neu1, and the enzymatically inactive splice variant of GAL form the elastin-binding protein complex. In humans, genetic defects of CathA cause Galactosialidosis, a metabolic disease characterized by combined deficiency of CathA, GAL, and Neu1 and a lysosomal storage of sialylated glycoconjugates. However, several phenotypic features of Galactosialidosis patients, including hypertension and cardiomyopathies, cannot be explained by the lysosomal storage. These observations suggest that CathA may be involved in hemodynamic functions that go beyond its protective activity in the lysosome. METHODS AND RESULTS: We generated a gene-targeted mouse in which the active CathA was replaced with a mutant enzyme carrying a Ser190Ala substitution in the active site. These animals expressed physiological amounts of catalytically inactive CathA protein, capable of forming lysosomal multienzyme complex, and did not develop secondary deficiency of Neu1 and GAL. Conversely, the mice showed a reduced degradation rate of the vasoconstrictor peptide, endothelin-1, and significantly increased arterial blood pressure. CathA-deficient mice also displayed scarcity of elastic fibers in lungs, aortic adventitia, and skin. CONCLUSIONS: Our results provide the first evidence that CathA acts in vivo as an endothelin-1-inactivating enzyme and strongly confirm a crucial role of this enzyme in effective elastic fiber formation.
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Enzymatic Activity of Lysosomal Carboxypeptidase (Cathepsin) A Is Required for Proper Elastic Fiber Formation and Inactivation of Endothelin-1
Circulation, 2008Co-Authors: Volkan Seyrantepe, Carlos R. Morales, Kohji Itoh, Yoshito Kadota, Aleksander Hinek, Junzheng Peng, Michael Fedjaev, Sheila Ernest, Maryssa Canuel, Julie L. LavoieAbstract:Background— Lysosomal carboxypeptidase, cathepsin A (protective protein, CathA), is a component of the lysosomal multienzyme complex along with β-galactosidase (GAL) and sialidase Neu1, where it activates Neu1 and protects GAL and Neu1 against the rapid proteolytic degradation. On the cell surface, CathA, Neu1, and the enzymatically inactive splice variant of GAL form the elastin-binding protein complex. In humans, genetic defects of CathA cause Galactosialidosis, a metabolic disease characterized by combined deficiency of CathA, GAL, and Neu1 and a lysosomal storage of sialylated glycoconjugates. However, several phenotypic features of Galactosialidosis patients, including hypertension and cardiomyopathies, cannot be explained by the lysosomal storage. These observations suggest that CathA may be involved in hemodynamic functions that go beyond its protective activity in the lysosome. Methods and Results— We generated a gene-targeted mouse in which the active CathA was replaced with a mutant enzyme carry...
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Elimination of abnormal sialylglycoproteins in fibroblasts with sialidosis and Galactosialidosis by normal gene transfer and enzyme replacement
Glycobiology, 2005Co-Authors: Yukako Oheda, Mai Murata, Yoshito Kadota, Masaharu Kotani, Yutaka Tatano, Jun Kuwahara, Hitoshi Sakuraba, Kohji Itoh, Yoshito Kadota, Kohji ItohAbstract:Sialidosis and Galactosialidosis are lysosomal storage diseases caused by the genetic defects of lysosomal sialidase (neuraminidase-1; NEU1) and lysosomal protective protein/cathepsin A (PPCA), respectively, associated with a NEU1 deficiency, excessive accumulation of sialylglycoconjugates, and development of progressive neurosomatic manifestations; in addition, the latter disorder is accompanied by simultaneous deficiencies of {szligbeta}-galactosidase and cathepsin A. We demonstrated that a few soluble N-glycosylated proteins carrying sialyloligosaccharides sensitive to glycopeptidase F (GPF) can be specifically detected in cultured fibroblasts from sialidosis and Galactosialidosis cases by blotting with a Maackia amurensis (MAM) lectin. We also examined the therapeutic effects of normal gene transfer and enzyme replacement by evaluating the decreases in sialylglycoconjugates accumulated in fibroblasts with these NEU1 deficiencies. The specific N-glycosylated proteins detected on MAM lectin blotting as well as the granular lysosomal fluorescence due to an avidin-FITC/biotinylated MAM lectin conjugate in sialidosis and Galactosialidosis fibroblasts disappeared in parallel with the restoration of the intracellular NEU1 activity after transfection of the recombinant NEU1 fused to HA tag sequence and the wild-type PPCA cDNA as well as administration of the recombinant PPCA precursor protein. The detection method for the abnormal sialylglycoproteins in cultured cells involving MAM lectin was demonstrated to be useful not only for biochemical and diagnostic analyses of NEU1 deficiencies but also for therapeutic evaluation of these conditions.
Alessandra Dazzo - One of the best experts on this subject based on the ideXlab platform.
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emphysema in an adult with Galactosialidosis linked to a defect in primary elastic fiber assembly
Molecular Genetics and Metabolism, 2012Co-Authors: Anna Lehman, Alessandra Dazzo, Yvan Campos, Andre Mattman, Peter D Pare, Zheyuan Zong, Sandra Sirrs, Aleksander HinekAbstract:Abstract Galactosialidosis is a lysosomal storage disorder caused by loss of function of protective protein cathepsin A, which leads to secondary deficiencies of β-galactosidase and neuraminidase-1. Emphysema has not been previously reported as a possible complication of this disorder, but we now describe this condition in a 41-year-old, non-smoking male. Our patient did not display deficiency in α-1-antitrypsin, the most common cause of emphysema in non-smokers, which brings about disseminated elastolysis. We therefore hypothesized that loss of cathepsin A activity was responsible because of previously published evidence showing it is prerequisite for normal elastogenesis. We now present experimental evidence to support this theory by demonstrating impaired primary elastogenesis in cultures of dermal fibroblasts from our patient. The obtained data further endorse our previous finding that functional integrity of the cell surface-targeted molecular complex of cathepsin A, neuraminidase-1 and the elastin-binding protein (spliced variant of β-galactosidase) is prerequisite for the normal assembly of elastic fibers. Importantly, we also found that elastic fiber production was increased after exposure either to losartan, spironolactone, or dexamethasone. Of immediate clinical relevance, our data suggest that surviving patients with Galactosialidosis should have periodic assessment of their pulmonary function. We also encourage further experimental exploration of therapeutic potential of the afore-mentioned elastogenesis-stimulating drugs for the alleviation of pathological processes in Galactosialidosis that could be mechanistically linked to impaired deposition of elastic fibers.
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heterodimerization of the sialidase neu1 with the chaperone protective protein cathepsin a prevents its premature oligomerization
Journal of Biological Chemistry, 2009Co-Authors: Erik Bonten, Brett Waddell, Valentin Zaitsev, Amanda Nourse, G L Taylor, Yvan Campos, William Lewis, Alessandra DazzoAbstract:Abstract Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with β-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive. Here, we have analyzed the hydrodynamic properties of PPCA, NEU1, and a complex of the two proteins and identified multiple binding sites on both proteins. One of these sites on NEU1 that is involved in binding to PPCA can also bind to other NEU1 molecules, albeit with lower affinity. Therefore, in the absence of PPCA, as in the lysosomal storage disease Galactosialidosis, NEU1 self-associates into chain-like oligomers. Binding of PPCA can reverse self-association of NEU1 by causing the disassembly of NEU1-oligomers and the formation of a PPCA-NEU1 heterodimeric complex. The identification of binding sites between the two proteins allowed us to create innovative structural models of the NEU1 oligomer and the PPCA-NEU1 heterodimeric complex. The proposed mechanism of interaction between NEU1 and its accessory protein PPCA provides a rationale for the secondary deficiency of NEU1 in Galactosialidosis.
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new mutations in two dutch patients with early infantile Galactosialidosis
Molecular Genetics and Metabolism, 2003Co-Authors: Johanna E M Groener, Yvan Campos, P Maaswinkelmooy, V Smit, M V D Hoeven, Jaap A Bakker, Alessandra DazzoAbstract:Abstract Galactosialidosis is an autosomal recessive lysosomal storage disease caused by a combined deficiency of lysosomal β-galactosidase and neuraminidase as a result of a primary defect in the protective protein/cathepsin A (PPCA). We report the first 2 Dutch cases of early infantile Galactosialidosis, both presenting with neonatal ascites. The defect was identified in urine, leukocytes, and fibroblasts. Residual activity was determined with a modified assay for cathepsin A and was PPCA transcript, which nevertheless had the correct size of 2 kb. Mutation analysis of both mRNA and genomic DNA from the patients identified two novel mutations in the PPCA locus. Case 1 was a compound heterozygote, with a single missense mutation in one allele, which resulted in Gly57Ser amino acid substitution, and a single C insertion at nucleotide position 899 in the second allele, which gave rise to a frame shift and premature termination codon. Case 2 was homozygous for the same C899 insertion found in case 1.
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the atomic model of the human protective protein cathepsin a suggests a structural basis for Galactosialidosis
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Erik Bonten, Gabrielle Rudenko, Alessandra DazzoAbstract:Human protective protein/cathepsin A (PPCA), a serine carboxypeptidase, forms a multienzyme complex with β-galactosidase and neuraminidase and is required for the intralysosomal activity and stability of these two glycosidases. Genetic lesions in PPCA lead to a deficiency of β-galactosidase and neuraminidase that is manifest as the autosomal recessive lysosomal storage disorder Galactosialidosis. Eleven amino acid substitutions identified in mutant PPCAs from clinically different Galactosialidosis patients have now been modeled in the three-dimensional structure of the wild-type enzyme. Of these substitutions, 9 are located in positions likely to alter drastically the folding and stability of the variant protein. In contrast, the other 2 mutations that are associated with a more moderate clinical outcome and are characterized by residual mature protein appeared to have a milder effect on protein structure. Remarkably, none of the mutations occurred in the active site or at the protein surface, which would have disrupted the catalytic activity or protective function. Instead, analysis of the 11 mutations revealed a substantive correlation between the effect of the amino acid substitution on the integrity of protein structure and the general severity of the clinical phenotype. The high incidence of PPCA folding mutants in Galactosialidosis reflects the fact that a single point mutation is unlikely to affect both the β-galactosidase and the neuraminidase binding sites of PPCA at the same time to produce the double glycosidase deficiency. Mutations in PPCA that result in defective folding, however, disrupt every function of PPCA simultaneously.
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early infantile Galactosialidosis clinical biochemical and molecular observations in a new patient
American Journal of Medical Genetics, 1996Co-Authors: Enrico Zammarchi, Xiao Yan Zhou, Maria Alice Donati, Amelia Morrone, Gianpaolo Donzelli, Alessandra DazzoAbstract:Few patients with the early-infantile form of Galactosialidosis have been described to date. Presented here is the first Italian case. Fetal hydrops was detected by ultrasound at week 24 of gestation. At birth, the infant presented with hypotonial, massive edema, a flattened coarse facies. telangiectasias, and hepatosplenomegaly, but no dysostosis multiplex. The patient died 72 days postpartum. Excessive sialyloligosaccharides in urine, as well as vacuolation of lymphocytes and eosinophilic granulocytes in peripheral blood, were indicative of a lysosomal storage disease. In the patient`s fibroblasts, both {alpha}-neuraminidase and {beta}-galactosidase activities were severely reduced, and cathepsin A activity was <1% of control levels, confirming the biochemical diagnosis of Galactosialidosis. However, in contrast to previously reported early-infantile cases, a normal amount of protective protein/cathepsin A mRNA was detected on Northern blots. This mutant transcript was translated into a precursor protein that was not processed into the mature enzyme and lacked both protective and catalytic activities. 28 refs., 4 figs., 1 tab.
Alexey V. Pshezhetsky - One of the best experts on this subject based on the ideXlab platform.
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Serine carboxypeptidases in regulation of vasoconstriction and elastogenesis.
Trends in Cardiovascular Medicine, 2020Co-Authors: Alexey V. Pshezhetsky, Aleksander Hinek, Aleksander HinekAbstract:Lysosomal carboxypeptidases play important roles in catabolism of proteins and peptides and in posttranslational processing of other lysosomal enzymes. The major lysosomal serine carboxypeptidase A (cathepsin A [CathA]), also known as protective protein, activates and stabilizes two other lysosomal enzymes, β -galactosidase and neuraminidase/sialidase 1. Genetic deficiency of CathA (Galactosialidosis) causes the lysosomal storage of sialylated glycoconjugates and leads to a multiorgan pathology. The Galactosialidosis patients also show arterial hypertension and cardiomyopathy, conditions not predicted from the lysosomal storage of glycoconjugates. This review summarizes the experimental data suggesting that both cardiovascular pathologies associate with persisted vasoconstrictions and impaired formation of the elastic fibers triggered by the deficiency of CathA. We also discuss the homologous serine carboxypeptidases, Scpep1 and vitellogenic-like carboxypeptidase, that are secreted from endothelial cells and could potentially affect the cardiovascular system.
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Neu4, a Novel Human Lysosomal Lumen Sialidase, Confers Normal Phenotype to Sialidosis and Galactosialidosis Cells
Journal of Biological Chemistry, 2004Co-Authors: Volkan Seyrantepe, Karine Landry, Jacob A. Hassan, Stephanie Trudel, Carlos R. Morales, Alexey V. PshezhetskyAbstract:Abstract Three different mammalian sialidases have been described as follows: lysosomal (Neu1, gene NEU1), cytoplasmic (Neu2, gene NEU2), and plasma membrane (Neu3, gene NEU3). Because of mutations in the NEU1 gene, the inherited deficiency of Neu1 in humans causes the severe multisystemic neurodegenerative disorder sialidosis. Galactosialidosis, a clinically similar disorder, is caused by the secondary Neu1 deficiency because of genetic defects in cathepsin A that form a complex with Neu1 and activate it. In this study we describe a novel lysosomal lumen sialidase encoded by the NEU4 gene on human chromosome 2. We demonstrate that Neu4 is ubiquitously expressed in human tissues and has broad substrate specificity by being active against sialylated oligosaccharides, glycoproteins, and gangliosides. In contrast to Neu1, Neu4 is targeted to lysosomes by the mannose 6-phospate receptor and does not require association with other proteins for enzymatic activity. Expression of Neu4 in the cells of sialidosis and Galactosialidosis patients results in clearance of storage materials from lysosomes suggesting that Neu4 may be useful for developing new therapies for these conditions.
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molecular mechanism of lysosomal sialidase deficiency in Galactosialidosis involves its rapid degradation
Biochemical Journal, 1998Co-Authors: Maia V Vinogradova, Carlos R. Morales, Lorraine Michaud, Alexander V Mezentsev, Kiven E Lukong, Mohamed Elalfy, Michel Potier, Alexey V. PshezhetskyAbstract:: Galactosialidosis is an inherited lysosomal storage disease caused by the combined deficiency of lysosomal sialidase and beta-galactosidase secondary to the deficiency of cathepsin A/protective protein, which is associated with sialidase and beta-galactosidase in a high-molecular weight (1.27MDa) complex. Clinical phenotypes of patients as well as the composition of compounds which are stored in patient's tissues implicate sialidase deficiency as the underlying pathogenic defect. The recent cloning and sequencing of lysosomal sialidase [Pshezhetsky, Richard, Michaud, Igdoura, Wang, Elsliger, Qu, Leclerc, Gravel, Dallaire and Potier (1997), Nature Genet. 15, 316-320] allowed us to study the molecular mechanism of sialidase deficiency in Galactosialidosis. By Western blotting, using antibodies against the recombinant human enzyme, and by NH2-terminal sequencing, we showed that sialidase is synthesized as a 45.5 kDa precursor and after the cleavage of the 47-amino acid signal peptide and glycosylation becomes a 48.3 kDa mature active enzyme present in the 1.27 kDa complex. Transgenic expression of sialidase in cultured skin fibroblasts from normal controls and from Galactosialidosis patients, followed by immunofluorescent and immunoelectron microscopy showed that in both normal and affected cells the expressed sialidase was localized on lysosomal and plasma membranes, but the amount of sialidase found in Galactosialidosis cells was approximately 5-fold reduced. Metabolic labelling studies demonstrated that the 48.3 kDa mature active form of sialidase was stable in normal fibroblasts (half-life approximately 2.7 h), whereas in Galactosialidosis fibroblasts the enzyme was rapidly converted (half-life approximately 30 min) into 38.7 and 24 kDa catalytically inactive forms. Altogether our data provide evidence that the molecular mechanism of sialidase deficiency in Galactosialidosis is associated with abnormal proteolytic cleavage and fast degradation.
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molecular pathology of Galactosialidosis in a patient affected with two new frameshift mutations in the cathepsin a protective protein gene
Human Mutation, 1998Co-Authors: Catherine Richard, Michel Potier, Julie Tranchemontagne, Marcandre Elsliger, Grant A Mitchell, Alexey V. PshezhetskyAbstract:Galactosialidosis is a recessively inherited lysosomal storage disease characterized by the combined deficiency of neuraminidase and β-galactosidase secondary to the genetic deficiency of cathepsin A/protective protein. In lysosomes, cathepsin A forms a high-molecular-weight complex with β-galactosidase and neuraminidase that protects these enzymes against intralysosomal proteolysis. In a patient affected with late infantile form of Galactosialidosis, we found two new cathepsin A mutations, a two-nucleotide deletion, c517delTT and an intronic mutation, IVS8+9CG resulting in abnormal splicing and a five-nucleotide insertion in the cathepsin A cDNA. Both mutations cause frameshifts and result in the synthesis of truncated cathepsin A proteins, which, as suggested by structural modeling, are incapable of dimerization, complex formation, and catalysis. However, enzymatic assays, gel-filtration, and Western blot analysis of the patient's cultured skin fibroblast extracts showed the presence of a small amount of normal-size, catalytically active cathepsin A and cathepsin A-β-galactosidase 680 kDa complex, suggesting that a low amount of cathepsin A mRNA is spliced normally and produces the wild-type protein. This may contribute to the relatively mild phenotype of the patient and illustrates the importance of critically comparing molecular results with clinical and biochemical phenotypes. Hum Mutat 11:461–469, 1998. © 1998 Wiley-Liss, Inc.
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association of n acetylgalactosamine 6 sulfate sulfatase with the multienzyme lysosomal complex of β galactosidase cathepsin a and neuraminidase possible implication for intralysosomal catabolism of keratan sulfate
Journal of Biological Chemistry, 1996Co-Authors: Alexey V. Pshezhetsky, Michel PotierAbstract:Abstract N-Acetylgalactosamine-6-sulfate sulfatase (GALNS) catalyzes the first step of intralysosomal keratan sulfate (KS) catabolism. In Morquio type A syndrome GALNS deficiency causes the accumulation of KS in tissues and results in generalized skeletal dysplasia in affected patients. We show that in normal cells GALNS is in a 1.27-MDa complex with three other lysosomal hydrolases: β-galactosidase, α-neuraminidase, and cathepsin A (protective protein). GALNS copurifies with the complex by different chromatography techniques: affinity chromatography on both cathepsin A-binding and β-galactosidase-binding columns, gel filtration, and chromatofocusing. Anti-human cathepsin A rabbit antiserum coprecipitates GALNS together with cathepsin A, β-galactosidase, and α-neuraminidase in both a purified preparation of the 1.27-MDa complex and crude glycoprotein fraction from human placenta extract. Gel filtration analysis of fibroblast extracts of patients deficient in either β-galactosidase (β-galactosidosis) or cathepsin A (Galactosialidosis), which accumulate KS, demonstrates that the 1.27-MDa complex is disrupted and that GALNS is present only in free homodimeric form. The GALNS activity and cross-reacting material are reduced in the fibroblasts of patients affected with Galactosialidosis, indicating that the complex with cathepsin A may protect GALNS in the lysosome. We suggest that the 1.27-MDa complex of lysosomal hydrolases is essential for KS catabolism and that the disruption of this complex may be responsible for the KS accumulation in β-galactosidosis and Galactosialidosis patients.
Yoshiyuki Suzuki - One of the best experts on this subject based on the ideXlab platform.
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chemical chaperone treatment for Galactosialidosis effect of noev on β galactosidase activities in fibroblasts
Brain & Development, 2016Co-Authors: Mohammad Arif Hossain, Yoshiyuki Suzuki, Katsumi Higaki, Michiko Shinpo, Eiji Nanba, Keiichi Ozono, Norio SakaiAbstract:Abstract Introduction Galactosialidosis is a rare lysosomal storage disease caused by a combined deficiency of G M1 β-galactosidase (β-gal) and neuraminidase secondary to a defect of a lysosomal enzyme protective protein/cathepsin A (PPCA) and mutation in CTSA gene. Three subtypes are recognized: early infantile, late infantile, and juvenile/adult. There is no specific therapy for patients with Galactosialidosis at this time. Objectives The aim of this study was to determine the chaperone effect of N -octyl-4-epi-β-valienamine (NOEV) on β-gal proteins in skin fibroblasts of PPCA-deficit patients. Methods β-Gal and neuraminidase activities were measured for the diagnosis of the patients with Galactosialidosis. Western blotting for PPCA protein and direct sequencing for CTSA gene were performed. Cultured skin fibroblast were treated with NOEV. Results We report four novel patients with Galactosialidosis: one had the early infantile form and the other three had the juvenile/adult form. We found that NOEV stabilized β-gal activity in lysate from cultured skin fibroblasts from these patients. Treatment with NOEV significantly enhanced β-gal activity in cultured skin fibroblasts in the absence of PPCA. Conclusions Our results indicate the possibility that NOEV chaperone therapy might have a beneficial effect, at least in part, for patients with Galactosialidosis.
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Expression of protective protein in human tissue.
Pediatric Neurology, 1999Co-Authors: Osamu Sohma, Akira Satake, Masashi Mizuguchi, Hitoshi Sakuraba, Kohji Itoh, Yoshiyuki Suzuki, Sachio Takashima, Kohji Itoh, Kiyomitsu OyanagiAbstract:The authors investigated by immunohistochemistry the distribution of protective protein in human tissues. Immunoreactivity was observed in the cytoplasm, revealing a granular pattern and cell type specificity. The most intense staining was observed in the large neurons of brain, distal and collecting tubular cells of kidney, epithelial cells of bronchus, and Leydig cells of testis. In a patient with Galactosialidosis type IIa, all these stains were absent. The neurons that were most strongly stained in the control group, such as the Betz cells, neurons in the basal forebrain, motor neurons in the cranial nerve nuclei, and ventral horn cells of the spinal cord, were markedly ballooned in the patient with Galactosialidosis.
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molecular form and subcellular distribution of acid β galactosidase in fibroblasts from patients with gm1 gangliosidosis morquio b disease and Galactosialidosis
Brain & Development, 1997Co-Authors: Nobuaki Takiyama, Hitoshi Sakuraba, Kohji Itoh, Michie Shimmoto, J Nishimoto, Koji Inui, Yoshiyuki SuzukiAbstract:Abstract The molecular form and subcellular distribution of acid β-galactosidase in cultured fibroblasts from patients with β-galactosidase deficiency (G M1 -gangliosidosis, Morquio B disease and Galactosialidosis) were studied, using antibodies against three different forms of the human enzyme: a high-molecular-weight multienzymic complex, a recombinant 84-kDa precursor, and a 64-kDa tryptic product of the precursor. The mature enzyme from normal fibroblasts was immunoprecipitated by the anti-complex and anti-64-kDa protein antibodies, but not by the anti-84-kDa precursor one. Immunofluorescence staining of normal fibroblasts revealed the granular (lysosomal) distribution with anti-64-kDa protein antibody and the perinuclear reticular distribution with anti-84-kDa precursor antibody, probably representing the Golgi apparatus. Both patterns were demonstrated in Morquio B disease, but the residual enzyme activity was exclusively due to the mature enzyme. In Type 1 Galactosialidosis, most of the expressed enzyme was detected as the precursor form with a perinuclear reticular distribution. In Type 2 Galactosialidosis, more than half of the enzyme activity was due to the mature form with a lysosomal distribution. Fibroblasts from a patient with G M1 gangliosidosis, expressing no β-galactosidase mRNA, did not react against either anti-64-kDa protein antibody or anti-84-kDa precursor antibody. The combined use of immunoprecipitation and immunostaining was useful for analysing the pathophysiology of the intracellular processing and transport of the mutant β-galactosidase.
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Acid carboxypeptidase deficiency in Galactosialidosis
Japanese journal of human genetics, 1991Co-Authors: Kohji Itoh, Hitoshi Sakuraba, Michel Potier, Nobuaki Takiyama, Akihiro Oshima, Yoshiro Nagao, Yoshiyuki SuzukiAbstract:Carboxypeptidase activity with an optimal pH at 5.7 was found to be deficient in cultured lymphoblastoid cells and skin fibroblasts from 16 Galactosialidosis patients of Japanese origin. The amounts of residual enzyme activities did not correlate with clinical phenotypes (early infantile and juvenile/adult). Four parents of the patients from different families showed enzyme activities at an intermediate level between the patients and normal controls. It was concluded that this enzyme deficiency is closely connected to the genetic defect of “protective protein.” Further characterization with various protease inhibitors indicated that the enzyme deficient in Galactosialidosis cells is a serine carboxypeptidase with histidine and cysteine residues at or near the active site.
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Galactosialidosis simultaneous deficiency of esterase carboxy terminal deamidase and acid carboxypeptidase activities
Biochemical and Biophysical Research Communications, 1990Co-Authors: Ryoichi Kase, Hitoshi Sakuraba, Kohji Itoh, Nobuaki Takiyama, Akihiro Oshima, Yoshiyuki SuzukiAbstract:Abstract Esterase and deamidase activities at pH 7.0 and carboxypeptidase activity at pH 5.7 were markedly low or deficient in seven Galactosialidosis fibroblast strains with deficient activity of “protective protein” for lysosomal β-galactosidase and neuraminidase. No simultaneous deficiency of these three enzyme activities was observed in other lysosomal disease fibroblasts examined in this study. This result strongly suggests that “protective protein” is identical with a multifunctional protein with esterase/deamidase/carboxypeptidase activities and its mutation in Galactosialidosis results in deficiency of these three enzyme activities.
