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Acid Hydrolases

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Stuart Kornfeld – One of the best experts on this subject based on the ideXlab platform.

  • the dmap interaction domain of udp glcnac lysosomal enzyme n acetylglucosamine 1 phosphotransferase is a substrate recognition module
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Yi Qian, Heather Flanagansteet, Eline Van Meel, Richard Steet, Stuart Kornfeld
    Abstract:

    UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an α2β2γ2 heterohexamer that mediates the initial step in the formation of the mannose 6-phosphate recognition signal on lysosomal Acid Hydrolases. We previously reported that the specificity of the reaction is determined by the ability of the α/β subunits to recognize a conformation-dependent protein determinant present on the Acid Hydrolases. We now present evidence that the DNA methyltransferase-associated protein (DMAP) interaction domain of the α subunit functions in this recognition process. First, GST-DMAP pulled down several Acid Hydrolases, but not nonlysosomal glycoproteins. Second, recombinant GlcNAc-1-phosphotransferase containing a missense mutation in the DMAP interaction domain (Lys732Asn) identified in a patient with mucolipidosis II exhibited full activity toward the simple sugar α-methyl d-mannoside but impaired phosphorylation of Acid Hydrolases. Finally, unlike the WT enzyme, expression of the K732N mutant in a zebrafish model of mucolipidosis II failed to correct the phenotypic abnormalities. These results indicate that the DMAP interaction domain of the α subunit functions in the selective recognition of Acid hydrolase substrates and provides an explanation for the impaired phosphorylation of Acid Hydrolases in a patient with mucolipidosis II.

  • functions of the α β and γ subunits of udp glcnac lysosomal enzyme n acetylglucosamine 1 phosphotransferase
    Journal of Biological Chemistry, 2010
    Co-Authors: Yi Qian, Intaek Lee, Wang Sik Lee, Meiqian Qian, Mariko Kudo, William M Canfield, Peter Lobel, Stuart Kornfeld
    Abstract:

    Abstract UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α2β2γ2 hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal Acid Hydrolases. Using a multifaceted approach, including analysis of Acid hydrolase phosphorylation in mice and fibroblasts lacking the γ subunit along with kinetic studies of recombinant α2β2γ2 and α2β2 forms of the transferase, we have explored the function of the α/β and γ subunits. The findings demonstrate that the α/β subunits recognize the protein determinant of Acid Hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the α/β subunits phosphorylate about one-third of the Acid Hydrolases at close to wild-type levels but require the γ subunit for optimal phosphorylation of the rest of the Acid Hydrolases. In addition to enhancing the activity of the α/β subunits toward a subset of the Acid Hydrolases, the γ subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the γ subunit binds and presents the high mannose glycans of the acceptor to the α/β catalytic site in a favorable manner.

  • Murine UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase Lacking the γ-Subunit Retains Substantial Activity toward Acid Hydrolases
    Journal of Biological Chemistry, 2007
    Co-Authors: Bobby Joe Payne, Claire M. Gelfman, Peter Vogel, Stuart Kornfeld
    Abstract:

    Abstract UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on Acid Hydrolases. The transferase exists as anα2β2γ2 hexameric complex with the α- and β-subunits derived from a single precursor molecule. The catalytic function of the transferase is attributed to the α- and β-subunits, whereas the γ-subunit is believed to be involved in the recognition of a conformation-dependent protein determinant common to Acid Hydrolases. Using knock-out mice with mutations in either the α/β gene or the γ gene, we show that disruption of the α/β gene completely abolishes phosphorylation of high mannose oligosaccharides on Acid Hydrolases whereas knock-out of the γ gene results in only a partial loss of phosphorylation. These findings demonstrate that the α/β-subunits, in addition to their catalytic function, have some ability to recognize Acid Hydrolases as specific substrates. This process is enhanced by the γ-subunit.

Yi Qian – One of the best experts on this subject based on the ideXlab platform.

  • the dmap interaction domain of udp glcnac lysosomal enzyme n acetylglucosamine 1 phosphotransferase is a substrate recognition module
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Yi Qian, Heather Flanagansteet, Eline Van Meel, Richard Steet, Stuart Kornfeld
    Abstract:

    UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an α2β2γ2 heterohexamer that mediates the initial step in the formation of the mannose 6-phosphate recognition signal on lysosomal Acid Hydrolases. We previously reported that the specificity of the reaction is determined by the ability of the α/β subunits to recognize a conformation-dependent protein determinant present on the Acid Hydrolases. We now present evidence that the DNA methyltransferase-associated protein (DMAP) interaction domain of the α subunit functions in this recognition process. First, GST-DMAP pulled down several Acid Hydrolases, but not nonlysosomal glycoproteins. Second, recombinant GlcNAc-1-phosphotransferase containing a missense mutation in the DMAP interaction domain (Lys732Asn) identified in a patient with mucolipidosis II exhibited full activity toward the simple sugar α-methyl d-mannoside but impaired phosphorylation of Acid Hydrolases. Finally, unlike the WT enzyme, expression of the K732N mutant in a zebrafish model of mucolipidosis II failed to correct the phenotypic abnormalities. These results indicate that the DMAP interaction domain of the α subunit functions in the selective recognition of Acid hydrolase substrates and provides an explanation for the impaired phosphorylation of Acid Hydrolases in a patient with mucolipidosis II.

  • functions of the α β and γ subunits of udp glcnac lysosomal enzyme n acetylglucosamine 1 phosphotransferase
    Journal of Biological Chemistry, 2010
    Co-Authors: Yi Qian, Intaek Lee, Wang Sik Lee, Meiqian Qian, Mariko Kudo, William M Canfield, Peter Lobel, Stuart Kornfeld
    Abstract:

    Abstract UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α2β2γ2 hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal Acid Hydrolases. Using a multifaceted approach, including analysis of Acid hydrolase phosphorylation in mice and fibroblasts lacking the γ subunit along with kinetic studies of recombinant α2β2γ2 and α2β2 forms of the transferase, we have explored the function of the α/β and γ subunits. The findings demonstrate that the α/β subunits recognize the protein determinant of Acid Hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the α/β subunits phosphorylate about one-third of the Acid Hydrolases at close to wild-type levels but require the γ subunit for optimal phosphorylation of the rest of the Acid Hydrolases. In addition to enhancing the activity of the α/β subunits toward a subset of the Acid Hydrolases, the γ subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the γ subunit binds and presents the high mannose glycans of the acceptor to the α/β catalytic site in a favorable manner.

Bernard Hoflack – One of the best experts on this subject based on the ideXlab platform.

  • Proteomic Analysis of Lysosomal Acid Hydrolases Secreted by Osteoclasts Implications for Lytic Enzyme Transport and Bone Metabolism
    Molecular & Cellular Proteomics, 2005
    Co-Authors: Cornelia Czupalla, Hannu Mansukoski, Thilo Riedl, Dorothee Thiel, Eberhard Krause, Bernard Hoflack
    Abstract:

    Osteoclasts, the bone-digesting cells, are polarized cells that secrete Acid Hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of Acid Hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NFB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing Acid Hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of Acid Hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these Acid Hydrolases were secreted as proproteins. The expression of most of the identified Acid Hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of Acid Hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna. Molecular & Cellular Proteomics 5:134–143, 2006.

  • Proteomic Analysis of Lysosomal Acid Hydrolases Secreted by Osteoclasts Implications for Lytic Enzyme Transport and Bone Metabolism
    Molecular & cellular proteomics : MCP, 2005
    Co-Authors: Cornelia Czupalla, Hannu Mansukoski, Thilo Riedl, Dorothee Thiel, Eberhard Krause, Bernard Hoflack
    Abstract:

    Osteoclasts, the bone-digesting cells, are polarized cells that secrete Acid Hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of Acid Hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-kappaB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing Acid Hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of Acid Hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these Acid Hydrolases were secreted as proproteins. The expression of most of the identified Acid Hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of Acid Hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna.