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Karl Wah Keung Tsim - One of the best experts on this subject based on the ideXlab platform.
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the assembly of proline rich Membrane Anchor prima linked acetylcholinesterase enzyme glycosylation is required for enzymatic activity but not for oligomerization
Journal of Biological Chemistry, 2011Co-Authors: Vicky P Chen, Roy Chi Yan Choi, Wallace K B Chan, Wing K Leung, Gallant K L Chan, Karl Wah Keung TsimAbstract:Acetylcholinesterase (AChE) Anchors onto cell Membranes by a transMembrane protein PRiMA (proline-rich Membrane Anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal “t-peptide” in AChE catalytic subunit (AChET). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChET plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChET mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher Km value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma Membrane.
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a new variant of proline rich Membrane Anchor prima of acetylcholinesterase in chicken expression in different muscle fiber types
Neuroscience Letters, 2009Co-Authors: Mokka K W Mok, Vicky P Chen, Roy Chi Yan Choi, Ka Wing Leung, Heidi Qunhui Xie, Ava J Y Guo, Judy T T Zhu, Karl Wah Keung TsimAbstract:Abstract Proline-rich Membrane Anchor (PRiMA) is a molecule to organize acetylcholinesterase (AChE) into tetrameric globular form (G 4 ) that Anchors onto the plasma Membrane in brain and muscle. In mammal, PRiMA is encoded by a single gene with two splicing variants, PRiMA I and PRiMA II: PRiMA II is different to PRiMA I by its absence of a C-terminal cytoplasmic domain. The existence of these isoforms has not been revealed in avian specie. By using RT-PCR and bioinformatic analyses, two splicing variants of PRiMA were identified in chicken cerebrum. One variant contains very similar domains as compared to mammalian PRiMA I. The other variant, named as PRiMA II, has a very distinct cytoplasmic C-terminus of having 26 amino acids. Both forms of chicken PRiMA were able to organize the formation of G 4 AChE when that was over expressed together with AChE T subunit in cultured cells. The level of PRiMA mRNA, mainly PRiMA I, was higher in slow-twitch muscle than that of in fast-twitch muscle of chicken. This finding suggests that the muscle fiber type-specific expression of G 4 AChE in chicken could be a result of the different expression pattern of PRiMA in fast- and slow-twitch muscles.
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transcriptional regulation of proline rich Membrane Anchor prima of globular form acetylcholinesterase in neuron an inductive effect of neuron differentiation
Brain Research, 2009Co-Authors: Roy Chi Yan Choi, Vicky P Chen, Wing K Leung, Karl Wah Keung TsimAbstract:Abstract The transcriptional regulation of proline-rich Membrane Anchor (PRiMA), an Anchoring protein of tetrameric globular form of acetylcholinesterase (G4 AChE), was revealed in cultured cortical neurons during differentiation. The level of AChET protein, total enzymatic activity and the amount of G4 AChE were dramatically increased during the neuron differentiation. RT-PCR analyses revealed that the transcript encoding PRiMA was significantly up-regulated in the differentiated neurons. To investigate the transcriptional mechanism on PRiMA regulation, a reporter construct of human PRiMA promoter-tagged luciferase was employed in this study. Upon the neuronal differentiation in cortical neurons, a mitogen-activated protein (MAP) kinase-dependent pathway was stimulated: this signaling cascade was shown to regulate the transcriptional activity of PRiMA. In addition, both PRiMA and AChET transcripts were induced by the over expression of an active mutant of Raf in the cultured neurons. The treatment of a MAP kinase inhibitor (U0126) significantly blocked the expression of PRiMA transcript and promoter-driven luciferase activity as induced by the differentiation of cortical neurons. These results suggested that a MAP kinase signaling pathway served as one of the transcriptional regulators in controlling PRiMA gene expression during the neuronal differentiation process.
Michael A. J. Ferguson - One of the best experts on this subject based on the ideXlab platform.
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structure of the glycosylphosphatidylinositol Membrane Anchor glycan of a class 2 variant surface glycoprotein from trypanosoma brucei
Journal of Molecular Biology, 1998Co-Authors: Angela Mehlert, Julia M Richardson, Michael A. J. FergusonAbstract:Abstract The neutral glycan fraction of the glycosylphosphatidylinositol (GPI) Membrane Anchor of a class-2 variant surface glycoprotein (VSG) from Trypanosoma brucei was isolated following aqueous hydrogen fluoride dephosphorylation and nitrous acid deamination of the purified glycoprotein. The neutral glycans were fractionated by high-pH anion exchange chromatography and gel-filtration and six major glycan structures were solved by a combination of one and two-dimensional NMR, composition analysis, methylation linkage analysis and electrospray-mass spectrometry. The glycans were similar to those previously described for class-1 VSGs, in that they contained the linear trimannosyl sequence Manα1-2Manα1-6Man and a complex α-galactose branch of up to Galα1-2Galα1-6(Galα1-2)Gal, but most also contained an additional galactose residue attached α1-2 to the non-reducing terminal mannose residue and about one-third contained an additional galactose residue attached β1-3 to the middle mannose residue. The additional complexity of the class-2 VSG GPI glycans is discussed in terms of a biosynthetic model that explains the full range of mature GPI structures that can be expressed on different VSG classes by the same trypanosome clone.
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expression of a variant surface glycoprotein of trypanosoma gambiense in procyclic forms of trypanosoma brucei shows that the cell type dictates the nature of the glycosylphosphatidylinositol Membrane Anchor attached to the glycoprotein
Biochemical Journal, 1997Co-Authors: Francoise Paturiauxhanocq, Michael A. J. Ferguson, Luc Vanhamme, Sylvie Rolin, J Hanocqquertier, Nicole Zitzmann, Maurice Geuskens, Etienne PaysAbstract:Procyclic forms of Trypanosoma brucei have been genetically modified to express the major metacyclic variant surface glycoprotein (VSG variant AnTat 11.17) of Trypanosoma gambiense. The VSG is expressed in an intact Membrane-bound form that can be detected over the entire plasma Membrane, together with procyclin, and as a series of lower-molecular-mass fragments that are mostly soluble degradation products. The presence of degraded VSG in the cells and the culture medium suggests that VSG is not efficiently processed and/or efficiently folded when expressed in procyclic cells. The level of procyclin expressed on the surface of these cells is slightly reduced, although there is no difference in procyclin mRNA levels. The intact Membrane-bound form of the VSG is N-glycosylated with oligomannose structures and contains a glycosylphosphatidylinositol (GPI) Membrane Anchor that can be biosynthetically labelled with [3H]ethanolamine. The Anchor is sensitive to mammalian GPI-specific phospholipase D but, like the Anchor of procyclin, it is resistant to the action of bacterial phosphatidylinositol-specific phospholipase C. This pattern of phospholipase sensitivity suggests that the GPI Anchor acquired by VSG when expressed in procyclics is acylated on the inositol ring and therefore resembles a procyclic procyclin-type Anchor rather than a trypomastigote VSG-type Anchor with respect to the lipid structure. The VSG expressed in procyclics was sensitive to the action of a mixture of sialidase, beta-galactosidase and beta-hexosaminidase, suggesting that the VSG GPI Anchor also contains a sialylated polylactosamine side-chain modification similar to that described for procyclin. These results indicate that the nature of the protein expressed has little influence on the post-translational modifications performed in the secretory pathway of procyclic trypanosomes.
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structure of the glycosylphosphatidylinositol Membrane Anchor of human placental alkaline phosphatase
Biochemical Journal, 1994Co-Authors: Christopher A Redman, Jane Thomasoates, Shigenori Ogata, Yukio Ikehara, Michael A. J. FergusonAbstract:The glycosylphosphatidylinositol Membrane Anchor of human placental alkaline phosphatase was isolated by exhaustive proteolysis followed by hydrophobic interaction chromatography. The resulting glycosylphosphatidylinositol-peptide was subjected to compositional analysis and chemical and enzymic modifications. The neutral-glycan fraction, prepared by dephosphorylation followed by HNO2 deamination and reduction, was sequenced using exoglycosidases and acetolysis. The phosphatidylinositol moiety was analysed by fast-atom bombardment mass spectrometry and gas chromatography-mass spectrometry. Taken together the data suggest the structure, Thr-Asp-ethanolamine-PO4-Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN-(sn-1-O- alkyl-2-O-acylglycerol-3-PO4-1-myo-D-inositol), which contains an additional ethanolamine phosphate group at an unknown position.
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structure of the glycosyl phosphatidylinositol Membrane Anchor of acetylcholinesterase from the electric organ of the electric fish torpedo californica
Biochemical Journal, 1993Co-Authors: Angela Mehlert, Israel Silman, L Varon, S W Homans, Michael A. J. FergusonAbstract:The structure of the glycan moiety of the glycosyl-phosphatidylinositol (GPI) Membrane Anchor from Torpedo californica (electric fish) electric-organ acetylcholinesterase was solved using n.m.r., methylation analysis and chemical and enzymic micro-sequencing. Two structures were found to be present: Glc alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol and Glc alpha 1-2Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI Anchor structure is a novel feature. The Anchor was also shown to contain 2.3 residues of ethanolamine per molecule.
Frank F Bier - One of the best experts on this subject based on the ideXlab platform.
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construction of an artificial cell Membrane Anchor using darc as a fitting for artificial extracellular functionalities of eukaryotic cells
Journal of Nanobiotechnology, 2012Co-Authors: Markus Von Nickischrosenegk, Till Teschke, Frank F BierAbstract:The need to functionalize cell Membranes in a directed way for specific applications as single cell arrays or to force close cell-to-cell contact for artificial intercellular interaction and/or induction concerning stem cell manipulation or in general to have a tool for Membrane and cell surface-associated processes, we envisaged a neutral inactive Membrane Anchor for extracellular entities to facillitate the above mentioned functionalities.
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Construction of an artificial cell Membrane Anchor using DARC as a fitting for artificial extracellular functionalities of eukaryotic cells
Journal of Nanobiotechnology, 2012Co-Authors: Markus Von Nickisch-rosenegk, Till Teschke, Frank F BierAbstract:The need to functionalize cell Membranes in a directed way for specific applications as single cell arrays or to force close cell-to-cell contact for artificial intercellular interaction and/or induction concerning stem cell manipulation or in general to have a tool for Membrane and cell surface-associated processes, we envisaged a neutral inactive Membrane Anchor for extracellular entities to facillitate the above mentioned functionalities. The silent Duffy antigen/receptor for chemokines (DARC) is a receptor-like Membrane protein of erythrocytes and mediates no cell transduction not at least regarding a missing or truncated G-loop and therefore it seemed to be the candidate for our cell Membrane Anchor. We isolated the genetic information of DARC from human genomic DNA and cloned it in a mammalian cell line as a fusion protein via a suitable plasmid vector. In this report we demonstrate that the human plasma Membrane protein DARC can be used as an artificial Anchor molecule in cell surface engineering applications. We constructed the fusion protein SNAP-tag-DARC, consisting of DARC and the self-labeling protein tag SNAP-tag^® (Covalys). The SNAP-tag® served as an example for a molecular-technological developed protein that is artificially attached to the extracellular side of the plasma Membrane through our DARC-Anchor. SnapTag should serve as an example for any extracellular entity and was easy to detect by a commercial detection system. The synthesis of SNAP-tag-DARC, its correct incorporation into the cell Membrane and the functionality of the SNAP-tag® were verified by RT-PCR, Western blotting and confocal fluorescence microscopy and showed the desired functionality as an Membrane Anchor for an extracellular application entity.
D. Moradpour - One of the best experts on this subject based on the ideXlab platform.
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nmr structure and molecular dynamics of the in plane Membrane Anchor of nonstructural protein 5a from bovine viral diarrhea virus
Biochemistry, 2006Co-Authors: N. Sapay, V. Brass, G. Deleage, D. Moradpour, Roland Montserret, Christophe Chipot, Francois PeninAbstract:Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a monotopic Membrane protein Anchored to the Membrane by an N-terminal in-plane amphipathic alpha-helix. This Membrane Anchor is essential for the assembly of a functional viral replication complex. Although amino acid sequences differ considerably, putative Membrane Anchors with amphipathic features were predicted in NS5A from related Flaviviridae family members, in particular bovine viral diarrhea virus (BVDV), the prototype representative of the genus Pestivirus. We report here the NMR structure of the Membrane Anchor 1-28 of NS5A from BVDV in the presence of different Membrane mimetic media. This Anchor includes a long amphipathic alpha-helix of 21 residues interacting in-plane with the Membrane interface and including a putative flexible region. Molecular dynamic simulation at a water-dodecane interface used to mimic the surface separating a lipid bilayer and an aqueous medium demonstrated the stability of the helix orientation and the location at the hydrophobic-hydrophilic interface. The flexible region of the helix appears to be required to allow the most favorable interaction of hydrophobic and hydrophilic side chain residues with their respective environment at the Membrane interface. Despite the lack of amino acid sequence similarity, this amphipathic helix shares common structural features with that of the HCV counterpart, including a stable, hydrophobic N-terminal segment separated from the more hydrophilic C-terminal segment by a local, flexible region. These structural conservations point toward conserved roles of the N-terminal in-plane Membrane Anchors of NS5A in replication complex formation of HCV, BVDV, and other related viruses.
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structure and function of the Membrane Anchor domain of hepatitis c virus nonstructural protein 5a
Journal of Biological Chemistry, 2004Co-Authors: Francois Penin, V. Brass, Nicole Appel, Stephanie Ramboarina, Roland Montserret, Damien Ficheux, Hubert E Blum, D. MoradpourAbstract:Abstract Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a Membrane-associated, essential component of the viral replication complex. Here, we report the three-dimensional structure of the Membrane Anchor domain of NS5A as determined by NMR spectroscopy. An α-helix extending from amino acid residue 5 to 25 was observed in the presence of different Membrane mimetic media. This helix exhibited a hydrophobic, Trprich side embedded in detergent micelles, while the polar, charged side was exposed to the solvent. Thus, the NS5A Membrane Anchor domain forms an in-plane amphipathic α-helix embedded in the cytosolic leaflet of the Membrane bilayer. Interestingly, mutations affecting the positioning of fully conserved residues located at the cytosolic surface of the helix impaired HCV RNA replication without interfering with the Membrane association of NS5A. In conclusion, the NS5A Membrane Anchor domain constitutes a unique platform that is likely involved in specific interactions essential for the assembly of the HCV replication complex and that may represent a novel target for antiviral intervention.
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an amino terminal amphipathic α helix mediates Membrane association of the hepatitis c virus nonstructural protein 5a
Journal of Biological Chemistry, 2002Co-Authors: V. Brass, Francois Penin, He Blum, Roland Montserret, Elke Bieck, Benno Wolk, J A Hellings, D. MoradpourAbstract:Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a Membrane-associated viral replication complex. The determinants for Membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the Membrane Anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral Membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A Membrane Anchor, designated NS5A(1–31), demonstrated the presence of an amphipathic α-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of Membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER Membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention.
Vicky P Chen - One of the best experts on this subject based on the ideXlab platform.
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the assembly of proline rich Membrane Anchor prima linked acetylcholinesterase enzyme glycosylation is required for enzymatic activity but not for oligomerization
Journal of Biological Chemistry, 2011Co-Authors: Vicky P Chen, Roy Chi Yan Choi, Wallace K B Chan, Wing K Leung, Gallant K L Chan, Karl Wah Keung TsimAbstract:Acetylcholinesterase (AChE) Anchors onto cell Membranes by a transMembrane protein PRiMA (proline-rich Membrane Anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal “t-peptide” in AChE catalytic subunit (AChET). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChET plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChET mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher Km value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma Membrane.
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a new variant of proline rich Membrane Anchor prima of acetylcholinesterase in chicken expression in different muscle fiber types
Neuroscience Letters, 2009Co-Authors: Mokka K W Mok, Vicky P Chen, Roy Chi Yan Choi, Ka Wing Leung, Heidi Qunhui Xie, Ava J Y Guo, Judy T T Zhu, Karl Wah Keung TsimAbstract:Abstract Proline-rich Membrane Anchor (PRiMA) is a molecule to organize acetylcholinesterase (AChE) into tetrameric globular form (G 4 ) that Anchors onto the plasma Membrane in brain and muscle. In mammal, PRiMA is encoded by a single gene with two splicing variants, PRiMA I and PRiMA II: PRiMA II is different to PRiMA I by its absence of a C-terminal cytoplasmic domain. The existence of these isoforms has not been revealed in avian specie. By using RT-PCR and bioinformatic analyses, two splicing variants of PRiMA were identified in chicken cerebrum. One variant contains very similar domains as compared to mammalian PRiMA I. The other variant, named as PRiMA II, has a very distinct cytoplasmic C-terminus of having 26 amino acids. Both forms of chicken PRiMA were able to organize the formation of G 4 AChE when that was over expressed together with AChE T subunit in cultured cells. The level of PRiMA mRNA, mainly PRiMA I, was higher in slow-twitch muscle than that of in fast-twitch muscle of chicken. This finding suggests that the muscle fiber type-specific expression of G 4 AChE in chicken could be a result of the different expression pattern of PRiMA in fast- and slow-twitch muscles.
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transcriptional regulation of proline rich Membrane Anchor prima of globular form acetylcholinesterase in neuron an inductive effect of neuron differentiation
Brain Research, 2009Co-Authors: Roy Chi Yan Choi, Vicky P Chen, Wing K Leung, Karl Wah Keung TsimAbstract:Abstract The transcriptional regulation of proline-rich Membrane Anchor (PRiMA), an Anchoring protein of tetrameric globular form of acetylcholinesterase (G4 AChE), was revealed in cultured cortical neurons during differentiation. The level of AChET protein, total enzymatic activity and the amount of G4 AChE were dramatically increased during the neuron differentiation. RT-PCR analyses revealed that the transcript encoding PRiMA was significantly up-regulated in the differentiated neurons. To investigate the transcriptional mechanism on PRiMA regulation, a reporter construct of human PRiMA promoter-tagged luciferase was employed in this study. Upon the neuronal differentiation in cortical neurons, a mitogen-activated protein (MAP) kinase-dependent pathway was stimulated: this signaling cascade was shown to regulate the transcriptional activity of PRiMA. In addition, both PRiMA and AChET transcripts were induced by the over expression of an active mutant of Raf in the cultured neurons. The treatment of a MAP kinase inhibitor (U0126) significantly blocked the expression of PRiMA transcript and promoter-driven luciferase activity as induced by the differentiation of cortical neurons. These results suggested that a MAP kinase signaling pathway served as one of the transcriptional regulators in controlling PRiMA gene expression during the neuronal differentiation process.