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Volkmar Gieselmann - One of the best experts on this subject based on the ideXlab platform.
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Specific hammerhead ribozymes reduce synthesis of cation-independent Mannose 6-Phosphate Receptor mRNA and protein
Gene Therapy, 2003Co-Authors: A Yaghootfam, Volkmar GieselmannAbstract:Storage diseases because of lysosomal enzyme deficiencies may be treated by the transplantation of cells that secrete the enzyme which is deficient in patients. One can expect that increasing the amount of secreted enzymes will improve the therapy efficacy. Secretion of lysosomal enzymes can be enhanced by reducing the Mannose 6-Phosphate Receptor involved in the lysosomal sorting of newly synthesized lysosomal enzymes. For this purpose, we have constructed hammerhead ribozymes targeting the mRNA of the large murine Mannose 6-Phosphate Receptor (M6PR300). In vitro ribozymes cleave M6PR300 RNA fragments efficiently with cleavage rates of 69–93% after 3 h of incubation. Ribozymes were cloned into an expression vector in which they are integrated into the VaI adenovirus RNA to increase stability and in which they are transcribed from an RNA polymerase III promoter. These plasmids were transiently transfected into BHK cells to investigate in vivo activity. Two ribozymes reduce efficiently the levels of murine M6PR300 mRNA in transient transfection experiments to 42–45%. This correlates with the reduction of M6PR300 biosynthesis, which is reduced also to 37% of normal. We can also demonstrate that the reduction in M6PR300 is sufficient to increase a lysosomal enzyme secretion.
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Specific hammerhead ribozymes reduce synthesis of cation-independent Mannose 6-Phosphate Receptor mRNA and protein.
Gene therapy, 2003Co-Authors: A Yaghootfam, Volkmar GieselmannAbstract:Specific hammerhead ribozymes reduce synthesis of cation-independent Mannose 6-Phosphate Receptor mRNA and protein
A Yaghootfam - One of the best experts on this subject based on the ideXlab platform.
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Specific hammerhead ribozymes reduce synthesis of cation-independent Mannose 6-Phosphate Receptor mRNA and protein
Gene Therapy, 2003Co-Authors: A Yaghootfam, Volkmar GieselmannAbstract:Storage diseases because of lysosomal enzyme deficiencies may be treated by the transplantation of cells that secrete the enzyme which is deficient in patients. One can expect that increasing the amount of secreted enzymes will improve the therapy efficacy. Secretion of lysosomal enzymes can be enhanced by reducing the Mannose 6-Phosphate Receptor involved in the lysosomal sorting of newly synthesized lysosomal enzymes. For this purpose, we have constructed hammerhead ribozymes targeting the mRNA of the large murine Mannose 6-Phosphate Receptor (M6PR300). In vitro ribozymes cleave M6PR300 RNA fragments efficiently with cleavage rates of 69–93% after 3 h of incubation. Ribozymes were cloned into an expression vector in which they are integrated into the VaI adenovirus RNA to increase stability and in which they are transcribed from an RNA polymerase III promoter. These plasmids were transiently transfected into BHK cells to investigate in vivo activity. Two ribozymes reduce efficiently the levels of murine M6PR300 mRNA in transient transfection experiments to 42–45%. This correlates with the reduction of M6PR300 biosynthesis, which is reduced also to 37% of normal. We can also demonstrate that the reduction in M6PR300 is sufficient to increase a lysosomal enzyme secretion.
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Specific hammerhead ribozymes reduce synthesis of cation-independent Mannose 6-Phosphate Receptor mRNA and protein.
Gene therapy, 2003Co-Authors: A Yaghootfam, Volkmar GieselmannAbstract:Specific hammerhead ribozymes reduce synthesis of cation-independent Mannose 6-Phosphate Receptor mRNA and protein
Kurt Von Figura - One of the best experts on this subject based on the ideXlab platform.
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Identification of the putative Mannose 6-Phosphate Receptor (MPR 46) protein in the invertebrate mollusc.
Bioscience reports, 2002Co-Authors: Siva Kumar Nadimpalli, Kurt Von FiguraAbstract:Mannose 6-Phosphate Receptor (MPR 300) protein was earlier affinity purified on phosphomannan gel from the membrane extracts of whole animal acetone powder of a mollusc, unio, in the presence of EDTA (Udaya Lakshmi, Y., Radha, Y., Hille-Rehfeld, A., von Figura, K., and Siva Kumar, N. (1999) Biosci. Rep. 19:403-409). In the present study we demonstrate that the unio also contains the putative Mannose 6-Phosphate Receptor (MPR 46) that can be purified on the same gel in presence of divalent metal ions (10 mM each of calcium, manganese, and magnesium), and in the absence of sodium chloride and at pH 6.5. Chicken and Fish cell MPR 46 proteins were purified under these conditions (Siva Kumar, N., Udaya Lakshmi, Y., Hille-Rehfeld, A., and von Figura, K. (1999) Comp. Biochem. & PhysioL 123B:261-265). The authenticity of the Receptor is further confirmed by its ability to react with the MSC1 antibody that is specific for MPR 46 protein. Additional evidence for the presence of MPR 46 in molluscs could be obtained by metabolic labeling of mollusc cells Biomphalaria glabrata (Bg cells) with [35S] methionine and cysteine, and passing the labeled membrane extract on phosphomannan gel (at pH 6.5 and 7.0). On elution with Mannose 6-Phosphate, followed by immunoprecipitation of the column fractions, we identified the putative MPR 46 protein in the Bg cells. When Bg cell MPR 46 was deglycosylated along with chicken MPR 46 (control) both species yielded a single polypeptide corresponding to molecular mass of 26 kDa, suggesting that both contain the same Receptor protein.
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Identification of the putative Mannose 6-Phosphate Receptor protein (MPR 300) in the invertebrate unio.
Bioscience reports, 1999Co-Authors: Yerramalla Udaya Lakshmi, Annette Hille-rehfeld, Kurt Von Figura, Yalamarthy Radha, Nadimpalli Siva KumarAbstract:In mammals, Mannose 6-Phosphate Receptor proteins (MPR 300 and MPR 46) mediate transport of lysosomal enzymes to lysosomes. Both Receptors have been found in non-mammalian vertebrates including fish. To investigate the presence of MPRs in invertebrates, MPR 300 protein was isolated from the mollusc unio by affinity chromatography. It was shown to exhibit biochemical and immunological properties similar to mammalian MPR 300.
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The Oligomeric State of 46‐kDa Mannose 6‐Phosphate Receptor does not Change Upon Intracellular Recycling and Binding of Ligands
FEBS Journal, 1996Co-Authors: Eeva‐liisa Punnonen, Tanja Wilke, Kurt Von Figura, Annette Hille-rehfeldAbstract:46-kDa Mannose-6-Phosphate Receptor forms homooligomers in cell membranes and in detergent solution. The quaternary structure of detergent-solubilized 46-kDa Mannose 6-Phosphate Receptor is regulated by the presence of ligands, pH and Receptor concentration [Waheed, A. & von Figura, K. (1990) Eur. J. Biochem. 193, 47–54). To find out whether the intracellular recycling of 46-kDa Mannose 6-Phosphate Receptor is accompanied by changes in its quaternary structure, we have performed chemical cross-linking in membranes of intact cells. In all conditions tested, the dimer was the predominating form (more than 67% of total 46-kDa Mannose 6-Phosphate Receptor). The amount of trimeric and tetrameric forms varied among cell lines and contributed up to 20 % of total endogenous 46-kDa Mannose 6-Phosphate Receptor in human and mouse fibroblasts. Within a given cell line, the ratio of the oligomers was not significantly changed upon elevating endosomal pH by bafilomycin A1, upon changes in Receptor occupancy (treatment of cells with tunicamycin or use of mouse fibroblasts deficient in 300-kDa Mannose 6-Phosphate Receptor), nor upon depletion of adaptors from clathrin-coated vesicles of the trans Golgi network by brefeldin A. At the cell surface, where 46-kDa Mannose 6-Phosphate Receptor does not bind ligands, the percentage of dimer was similar to that observed intracellularly. Thus, the oligomeric state of 46-kDa Mannose 6-Phosphate Receptor apparently does not change during recycling as well as binding and dissociation of ligands. In view of the abundance of the dimer of 46-kDa Mannose 6-Phosphate Receptor in situ, our data suggest that it represents the main physiologically active form of the Receptor, and therefore present indirect evidence that binding of ligands to 46-kDa Mannose 6-Phosphate Receptor is probably regulated by conformational changes of Receptor or ligand rather than by changes in the quaternary structure.
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The Oligomeric State of 46-kDa Mannose 6-Phosphate Receptor does not Change Upon Intracellular Recycling and Binding of Ligands
European journal of biochemistry, 1996Co-Authors: Eeva‐liisa Punnonen, Tanja Wilke, Kurt Von Figura, Annette Hille-rehfeldAbstract:46-kDa Mannose-6-Phosphate Receptor forms homooligomers in cell membranes and in detergent solution. The quaternary structure of detergent-solubilized 46-kDa Mannose 6-Phosphate Receptor is regulated by the presence of ligands, pH and Receptor concentration [Waheed, A. & von Figura, K. (1990) Eur. J. Biochem. 193, 47–54). To find out whether the intracellular recycling of 46-kDa Mannose 6-Phosphate Receptor is accompanied by changes in its quaternary structure, we have performed chemical cross-linking in membranes of intact cells. In all conditions tested, the dimer was the predominating form (more than 67% of total 46-kDa Mannose 6-Phosphate Receptor). The amount of trimeric and tetrameric forms varied among cell lines and contributed up to 20 % of total endogenous 46-kDa Mannose 6-Phosphate Receptor in human and mouse fibroblasts. Within a given cell line, the ratio of the oligomers was not significantly changed upon elevating endosomal pH by bafilomycin A1, upon changes in Receptor occupancy (treatment of cells with tunicamycin or use of mouse fibroblasts deficient in 300-kDa Mannose 6-Phosphate Receptor), nor upon depletion of adaptors from clathrin-coated vesicles of the trans Golgi network by brefeldin A. At the cell surface, where 46-kDa Mannose 6-Phosphate Receptor does not bind ligands, the percentage of dimer was similar to that observed intracellularly. Thus, the oligomeric state of 46-kDa Mannose 6-Phosphate Receptor apparently does not change during recycling as well as binding and dissociation of ligands. In view of the abundance of the dimer of 46-kDa Mannose 6-Phosphate Receptor in situ, our data suggest that it represents the main physiologically active form of the Receptor, and therefore present indirect evidence that binding of ligands to 46-kDa Mannose 6-Phosphate Receptor is probably regulated by conformational changes of Receptor or ligand rather than by changes in the quaternary structure.
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Isolation and analysis of the human 46-kDa Mannose 6-Phosphate Receptor gene.
European journal of biochemistry, 1991Co-Authors: Hans‐jürgen Klier, Kurt Von Figura, Regina PohlmannAbstract:From a genomic library in EMBL 3, two overlapping clones for the human 46-kDa Mannose 6-Phosphate Receptor (MPR46) were isolated, which span the entire coding sequence. The human MPR46 gene is distributed over 12 kb and is divided into seven exons (110-1573 bp). All the intron/exon borders agree with the consensus sequences of splice junctions. Exon 1 codes for a 5' untranslated sequence. The ATG initiation codon begins with the second nucleotide in exon 2. A signal sequence of 26 amino acid residues is followed by the extracytoplasmic (luminal) domain, which extends to exon 5. The transmembrane domain of the Receptor spans exons 5 and 6 and the cytoplasmic domain is encoded by exons 6 and 7. The latter domain also codes for an extended 3' untranslated sequence. The transcription-initiation site was defined by primer extension. The sequence upstream of the cap site has strong promoter activity and contains structural elements characteristic of promoters found in housekeeping genes. No correlation between the genomic organization and known protein domains of the MPR46 was apparent. Moreover, the sequence of about 150 amino acids within the luminal domain of MPR46, which is homologous to the 15 repeats that constitute the luminal domain of the 300-kDa Mannose 6-Phosphate Receptor (MPR300), does not correlate with intron/exon borders. MPR46 and MPR300 have therefore diverged from a common ancestral gene before introduction of the present intron sequences.
Siva Kumar Nadimpalli - One of the best experts on this subject based on the ideXlab platform.
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Molecular cloning, expression and functional characterization of the chicken cation dependent Mannose 6-Phosphate Receptor protein.
International journal of biological macromolecules, 2008Co-Authors: Praveen Kumar Amancha, Siva Kumar NadimpalliAbstract:Mammalian Mannose 6-Phosphate (M6P) Receptors function in transport of lysosomal enzymes. To understand the structural and functional significance of the chicken cation dependent Mannose 6-Phosphate Receptor (MPR) (Mr 46 kDa), a full-length cDNA for the chicken protein was cloned and expressed in mpr(-/-) MEF cells devoid of both the Receptors. The stably transfected cells express the Receptor that could be affinity purified by phosphomannan chromatography. The authenticity of the Receptor was confirmed by its immuno-reactivity with mammalian MPR 46 antibodies and its ability to sort cathepsin D in transfected cells (92.3%) as compared to mock transfected cells (50.2%), establishing a functional role for the chicken Receptor.
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Mannose 6-Phosphate Receptor (MPR 300) Proteins From Goat and Chicken Bind Human IGF-II
Bioscience reports, 2006Co-Authors: Suresh Koduru, Sivaramakrishna Yadavalli, Siva Kumar NadimpalliAbstract:Mannose 6-Phosphate Receptor proteins (MPR 300 and 46) in mammals have been shown to mediate transport of lysosomal enzymes to lysosomes intracellularly. Both Receptors are also expressed on the plasma membrane. Only MPR 300 protein on the plasma membrane has been shown to be a multifunctional protein which in addition to binding Mannose 6-Phosphate containing proteins also binds human insulin-like growth factor-II (IGF-II) causing its internalization [Hille-Rehfeld, A. (1995) Mannose 6-Phosphate Receptors in sorting and transport of lysosomal enzymes. Biochim. Biophys. Acta. 1241: 177–194]. This property has been shown to be exhibited by other mammalian Receptors but not by the chicken and frog Receptors. In a recent study however it was shown that the fish embryo MPR 300 binds human IGF-II. [Mendez, E., Planas, J.V., Castillo, J., Navarro, I. and Gutierrez, J. (2001) Identification of a type II insulin-like growth factor Receptor in fish embryos. Endocrinology, 142: 1090–1097]. In the present study, we demonstrate that the purified goat and chicken liver Receptors bind human IGF-II by employing cross-linking experiments (purified Receptors and radiolabeled IGF-II) and by ligand blotting (using purified Receptors and biotinylated IGF-II). Further CEF cells (chicken embryonic fibroblasts) that are known to contain the putative MPR 300 protein were employed to demonstrate that the CEF cell Receptor binds human IGF-II.
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an immuno affinity method for the purification of Mannose 6 phosphate Receptor proteins
Journal of Biochemical and Biophysical Methods, 2003Co-Authors: Koduru Suresh, Siva Kumar NadimpalliAbstract:In a recent study, we have developed an ELISA method to quantify the Mannose 6-Phosphate Receptor (MPR) proteins [J. Biochem. Biophys. Methods 52 (2002) 111]. In the present study, we have used the goat MPR 300 antibody and peptide specific antibodies to human MPR 46 to develop simple and efficient immuno-affinity matrices, which can be used to purify the MPR proteins from goat liver in a single step. The identity of the immuno-affinity purified Receptors is confirmed by their molecular masses as well as by their immunoreactivity.
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Identification of the putative Mannose 6-Phosphate Receptor (MPR 46) protein in the invertebrate mollusc.
Bioscience reports, 2002Co-Authors: Siva Kumar Nadimpalli, Kurt Von FiguraAbstract:Mannose 6-Phosphate Receptor (MPR 300) protein was earlier affinity purified on phosphomannan gel from the membrane extracts of whole animal acetone powder of a mollusc, unio, in the presence of EDTA (Udaya Lakshmi, Y., Radha, Y., Hille-Rehfeld, A., von Figura, K., and Siva Kumar, N. (1999) Biosci. Rep. 19:403-409). In the present study we demonstrate that the unio also contains the putative Mannose 6-Phosphate Receptor (MPR 46) that can be purified on the same gel in presence of divalent metal ions (10 mM each of calcium, manganese, and magnesium), and in the absence of sodium chloride and at pH 6.5. Chicken and Fish cell MPR 46 proteins were purified under these conditions (Siva Kumar, N., Udaya Lakshmi, Y., Hille-Rehfeld, A., and von Figura, K. (1999) Comp. Biochem. & PhysioL 123B:261-265). The authenticity of the Receptor is further confirmed by its ability to react with the MSC1 antibody that is specific for MPR 46 protein. Additional evidence for the presence of MPR 46 in molluscs could be obtained by metabolic labeling of mollusc cells Biomphalaria glabrata (Bg cells) with [35S] methionine and cysteine, and passing the labeled membrane extract on phosphomannan gel (at pH 6.5 and 7.0). On elution with Mannose 6-Phosphate, followed by immunoprecipitation of the column fractions, we identified the putative MPR 46 protein in the Bg cells. When Bg cell MPR 46 was deglycosylated along with chicken MPR 46 (control) both species yielded a single polypeptide corresponding to molecular mass of 26 kDa, suggesting that both contain the same Receptor protein.
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Antibodies against the cytoplasmic tail can differentiate between the quaternary forms of the Mr 46,000 Mannose 6-Phosphate Receptor.
FEBS letters, 1991Co-Authors: Siva Kumar Nadimpalli, Kurt Von Figura, Bernhard Schmidt, Annette HilleAbstract:An antiserum against a peptide of the cytoplasmic tail of the Mr 46,000 Mannose 6-Phosphate Receptor is described which recognizes preferentially the tetrameric versus the dimeric form of this Receptor. This indicates that the conformation of the cytoplasmic tail, which harbours signals necessary for the trafficking of the Receptor, depends on the quaternary structure of the Receptor.
Konrad Sandhoff - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis processing and targeting of sphingolipid activator protein sap precursor in cultured human fibroblasts Mannose 6 phosphate Receptor independent endocytosis of sap precursor
Journal of Biological Chemistry, 1996Co-Authors: Gabriele Vielhaber, Robert Hurwitz, Konrad SandhoffAbstract:Abstract Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on Mannose 6-Phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-Phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by Mannose 6-Phosphate Receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on Mannose 6-Phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.