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Akira Kurosaka - One of the best experts on this subject based on the ideXlab platform.
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a putative Polypeptide n acetylgalactosaminyltransferase williams beuren syndrome chromosome region 17 wbscr17 regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.
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A putative Polypeptide N-Acetylgalactosaminyltransferase/Williams-Beuren syndrome chromosome region 17 (WBSCR17) regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Sayoko Oki, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.
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Cloning and Expression of a Brain-Specific Putative UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase Gene
Biological & Pharmaceutical Bulletin, 2005Co-Authors: Naosuke Nakamura, Nobuyuki Itoh, Shinya Toba, Mitsuharu Hirai, Shinichi Morishita, Tadahisa Mikami, Morichika Konishi, Akira KurosakaAbstract:We isolated a rat cDNA clone and its human orthologue, which are most homologous to UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase 9, by homology-based PCR from brain. Nucleotide sequence analysis of these putative GalNAc-transferases (designated pt-GalNAc-T) showed that they contained structural features characteristic of the GalNAc-transferase family. It was also found that human pt-GalNAc-T was identical to the gene WBSCR17, which is reported to be in the critical region of patients with Williams-Beuren Syndrome, a neurodevelopmental disorder, and to be predominantly expressed in brain and heart. In order to investigate the expression of pt-GalNAc-T in brain in more detail, we first examined that of human pt-GalNAc-T by Northern blot analysis and found the expression of the 5.0-kb mRNA to be most abundant in cerebral cortex with somewhat less abundant in cellebellum. The expression of rat pt-GalNAc-T was investigated more extensively. The brain-specific expression of 2.0-kb and 5.0-kb transcripts was demonstrated by Northern blot analysis. In situ hybridization in the adult brain revealed high levels of expression in cerebellum, hippocampus, thalamus, and cerebral cortex. Moreover, observation at high magnification revealed the expression to be associated with neurons, but not with glial cells. Analysis of the rat embryos also demonstrated that rat pt-GalNAc-T was expressed in the nervous system, including in the diencephalons, cerebellar primordium, and dorsal root ganglion. However, recombinant human pt-GalNAc-T, which was expressed in insect cells, did not glycosylate several peptides derived from mammalian mucins, suggesting that it may have a strict substrate specificity. The brain-specific expression of pt-GalNAc-T suggested its involvement in brain development, through O-glycosylation of proteins in the neurons.
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Characterization of a novel Polypeptide N-Acetylgalactosaminyltransferase (dGalNAc-T3) from Drosophila.
Biological & Pharmaceutical Bulletin, 2004Co-Authors: Naosuke Nakamura, Shinya Toba, Kenji Katano, Akira KurosakaAbstract:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-transferases) catalyze the initial reaction of mucin-type O-glycosylation. Here, we report the first biochemical characterization of one of the Drosophila GalNAc-transferases, dGalNAc-T3. This enzyme retains conserved motifs essential for the catalytic activity, but is a novel isozyme in that it has several inserted sequences in its lectin-like domain. Northern hybridization analysis of this isozyme identified a 2.5-kb mRNA in Drosophila larva. Biochemical characterization was carried out using the recombinant soluble dGalNAc-T3 expressed in COS7 cells. dGalNAc-T3, which required Mn2+ for the activity, had a pH optimum ranging from pH 7.5 to 8.5, and glycosylated most effectively at 29—33 °C. Its Km for UDP-GalNAc was 10.7 μM, which is as low as that of mammalian isozymes. dGalNAc-T3 glycosylated the peptides containing a sequence of XTPXP or TTAAP most efficiently. The enzyme was irreversibly inhibited by p-chloromercuriphenylsulphonic acid, indicating the presence of essential Cys residues for the activity.
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Function of the lectin domain of Polypeptide N-Acetylgalactosaminyltransferase 1.
Biochemical and Biophysical Research Communications, 2002Co-Authors: Mari Tenno, Ferénc J Kézdy, Åke P. Elhammer, Akira KurosakaAbstract:Abstract All UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases cloned to date contain a lectin domain at the C-terminus, consisting of three tandem repeat sequences ( α , β , and γ). We previously reported that the α repeat of one of the most ubiquitous isozymes, GalNAc-T1, is a functional lectin that recognizes O-linked GalNAc residues on the acceptor Polypeptides with multiple acceptor sites; the domain appears not to be involved in the glycosylation of acceptors with a single acceptor site. In this report, we studied the function of the β and γ repeats in the GalNAc-T1 lectin domain, by site-directed mutagenesis and analysis of the catalytic properties of mutant enzymes. We found that the β repeat recognizes GalNAc and is involved in glycosylation of acceptors with multiple glycosylation sites. The γ repeat, on the other hand, showed no significant GalNAc-binding activity. These results indicate that the lectin domain of GalNAc-T1 has at least two functional repeats, allowing the possibility of multivalent interactions with GalNAc residues on the acceptor Polypeptide during glycosylation.
Yoshiaki Nakayama - One of the best experts on this subject based on the ideXlab platform.
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a putative Polypeptide n acetylgalactosaminyltransferase williams beuren syndrome chromosome region 17 wbscr17 regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.
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A putative Polypeptide N-Acetylgalactosaminyltransferase/Williams-Beuren syndrome chromosome region 17 (WBSCR17) regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Sayoko Oki, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.
Naosuke Nakamura - One of the best experts on this subject based on the ideXlab platform.
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a putative Polypeptide n acetylgalactosaminyltransferase williams beuren syndrome chromosome region 17 wbscr17 regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.
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A putative Polypeptide N-Acetylgalactosaminyltransferase/Williams-Beuren syndrome chromosome region 17 (WBSCR17) regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Sayoko Oki, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.
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Cloning and Expression of a Brain-Specific Putative UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase Gene
Biological & Pharmaceutical Bulletin, 2005Co-Authors: Naosuke Nakamura, Nobuyuki Itoh, Shinya Toba, Mitsuharu Hirai, Shinichi Morishita, Tadahisa Mikami, Morichika Konishi, Akira KurosakaAbstract:We isolated a rat cDNA clone and its human orthologue, which are most homologous to UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase 9, by homology-based PCR from brain. Nucleotide sequence analysis of these putative GalNAc-transferases (designated pt-GalNAc-T) showed that they contained structural features characteristic of the GalNAc-transferase family. It was also found that human pt-GalNAc-T was identical to the gene WBSCR17, which is reported to be in the critical region of patients with Williams-Beuren Syndrome, a neurodevelopmental disorder, and to be predominantly expressed in brain and heart. In order to investigate the expression of pt-GalNAc-T in brain in more detail, we first examined that of human pt-GalNAc-T by Northern blot analysis and found the expression of the 5.0-kb mRNA to be most abundant in cerebral cortex with somewhat less abundant in cellebellum. The expression of rat pt-GalNAc-T was investigated more extensively. The brain-specific expression of 2.0-kb and 5.0-kb transcripts was demonstrated by Northern blot analysis. In situ hybridization in the adult brain revealed high levels of expression in cerebellum, hippocampus, thalamus, and cerebral cortex. Moreover, observation at high magnification revealed the expression to be associated with neurons, but not with glial cells. Analysis of the rat embryos also demonstrated that rat pt-GalNAc-T was expressed in the nervous system, including in the diencephalons, cerebellar primordium, and dorsal root ganglion. However, recombinant human pt-GalNAc-T, which was expressed in insect cells, did not glycosylate several peptides derived from mammalian mucins, suggesting that it may have a strict substrate specificity. The brain-specific expression of pt-GalNAc-T suggested its involvement in brain development, through O-glycosylation of proteins in the neurons.
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Characterization of a novel Polypeptide N-Acetylgalactosaminyltransferase (dGalNAc-T3) from Drosophila.
Biological & Pharmaceutical Bulletin, 2004Co-Authors: Naosuke Nakamura, Shinya Toba, Kenji Katano, Akira KurosakaAbstract:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-transferases) catalyze the initial reaction of mucin-type O-glycosylation. Here, we report the first biochemical characterization of one of the Drosophila GalNAc-transferases, dGalNAc-T3. This enzyme retains conserved motifs essential for the catalytic activity, but is a novel isozyme in that it has several inserted sequences in its lectin-like domain. Northern hybridization analysis of this isozyme identified a 2.5-kb mRNA in Drosophila larva. Biochemical characterization was carried out using the recombinant soluble dGalNAc-T3 expressed in COS7 cells. dGalNAc-T3, which required Mn2+ for the activity, had a pH optimum ranging from pH 7.5 to 8.5, and glycosylated most effectively at 29—33 °C. Its Km for UDP-GalNAc was 10.7 μM, which is as low as that of mammalian isozymes. dGalNAc-T3 glycosylated the peptides containing a sequence of XTPXP or TTAAP most efficiently. The enzyme was irreversibly inhibited by p-chloromercuriphenylsulphonic acid, indicating the presence of essential Cys residues for the activity.
Nobuyuki Itoh - One of the best experts on this subject based on the ideXlab platform.
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a putative Polypeptide n acetylgalactosaminyltransferase williams beuren syndrome chromosome region 17 wbscr17 regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.
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A putative Polypeptide N-Acetylgalactosaminyltransferase/Williams-Beuren syndrome chromosome region 17 (WBSCR17) regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Sayoko Oki, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.
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Cloning and Expression of a Brain-Specific Putative UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase Gene
Biological & Pharmaceutical Bulletin, 2005Co-Authors: Naosuke Nakamura, Nobuyuki Itoh, Shinya Toba, Mitsuharu Hirai, Shinichi Morishita, Tadahisa Mikami, Morichika Konishi, Akira KurosakaAbstract:We isolated a rat cDNA clone and its human orthologue, which are most homologous to UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase 9, by homology-based PCR from brain. Nucleotide sequence analysis of these putative GalNAc-transferases (designated pt-GalNAc-T) showed that they contained structural features characteristic of the GalNAc-transferase family. It was also found that human pt-GalNAc-T was identical to the gene WBSCR17, which is reported to be in the critical region of patients with Williams-Beuren Syndrome, a neurodevelopmental disorder, and to be predominantly expressed in brain and heart. In order to investigate the expression of pt-GalNAc-T in brain in more detail, we first examined that of human pt-GalNAc-T by Northern blot analysis and found the expression of the 5.0-kb mRNA to be most abundant in cerebral cortex with somewhat less abundant in cellebellum. The expression of rat pt-GalNAc-T was investigated more extensively. The brain-specific expression of 2.0-kb and 5.0-kb transcripts was demonstrated by Northern blot analysis. In situ hybridization in the adult brain revealed high levels of expression in cerebellum, hippocampus, thalamus, and cerebral cortex. Moreover, observation at high magnification revealed the expression to be associated with neurons, but not with glial cells. Analysis of the rat embryos also demonstrated that rat pt-GalNAc-T was expressed in the nervous system, including in the diencephalons, cerebellar primordium, and dorsal root ganglion. However, recombinant human pt-GalNAc-T, which was expressed in insect cells, did not glycosylate several peptides derived from mammalian mucins, suggesting that it may have a strict substrate specificity. The brain-specific expression of pt-GalNAc-T suggested its involvement in brain development, through O-glycosylation of proteins in the neurons.
Yasushi Ishihama - One of the best experts on this subject based on the ideXlab platform.
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a putative Polypeptide n acetylgalactosaminyltransferase williams beuren syndrome chromosome region 17 wbscr17 regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.
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A putative Polypeptide N-Acetylgalactosaminyltransferase/Williams-Beuren syndrome chromosome region 17 (WBSCR17) regulates lamellipodium formation and macropinocytosis
Journal of Biological Chemistry, 2012Co-Authors: Yoshiaki Nakayama, Naosuke Nakamura, Masaki Wakabayashi, Yasushi Ishihama, Ayumi Miyake, Nobuyuki Itoh, Sayoko Oki, Akira KurosakaAbstract:Abstract We previously identified a novel Polypeptide N-Acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.