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Yasuhiro Kajihara – One of the best experts on this subject based on the ideXlab platform.

  • monitoring of Glycoprotein quality control system with a series of chemically synthesized homogeneous native and misfolded Glycoproteins
    Journal of the American Chemical Society, 2018
    Co-Authors: Tatsuto Kiuchi, Masayuki Izumi, Akira Seko, Masafumi Sakono, Ryo Okamoto, Yuki Mukogawa, Arisa Shimada, Yoichi Takeda, Yasuhiro Kajihara
    Abstract:

    The Glycoprotein quality control (GQC) system in the endoplasmic retireticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:Glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded Glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous Glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-β (IFN-β), and interleukin 8 (IL8) and their misfolded counterparts, and used these Glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chrochromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded Glycoproteins to native form through folding intermediates, allowing for the relationship between the amou…

  • chemical synthesis of homogeneous Glycoproteins for the study of Glycoprotein quality control system
    Israel Journal of Chemistry, 2015
    Co-Authors: Masayuki Izumi, Simone Dedola, Yasuhiro Kajihara
    Abstract:

    The Glycoprotein quality control system exists in the endoplasmic retireticulum to maintain protein homeostasis and prevent accumulation of aberrant Glycoproteins. Folding sensor enzyme uridine diphosphate (UDP)glucose : Glycoprotein glucosyltransferase (UGGT) plays an important role in this system through its ability to discriminate immature or misfolded Glycoproteins from native ones. UGGT transfers a glucose residue to a Glycoprotein containing Man9GlcNAc2 (M9; Man=mannose, GlcNAc=N-acetacetyllD-glucosamine) N-glycan only when the Glycoprotein has not attained a native form. We chemically prepared homogeneous Glycoproteins containing M9 N-glycan in the native form as well as in misfolded forms and examined them as substrates of UGGT. Glucose transfer to misfolded Glycoproteins was clearly observed by LC-MS, but Glycoproteins in the native form were barely glucosylated. Furthermore, we constructed an in vitro Glycoprotein folding system in the presence of UGGT and found out that all folding intermediates which appeared during folding were also glucosylated. Through these experiments, we demonstrated the usefulness of chemically synthesized homogeneous Glycoproteins as probes to gain insights into the molecular basis of the Glycoprotein quality control system.

  • Chemical Synthesis of Homogeneous Glycoproteins
    Glycoscience: Biology and Medicine, 2014
    Co-Authors: Masayuki Izumi, Ryo Okamoto, Yasuhiro Kajihara
    Abstract:

    Oligosaccharides of Glycoprotein are known to be heterogeneous. These diverse oligosaccharide profiles have been a hindrance to elucidating oligosaccharide functions in many biological events. In order to elucidate oligosaccharide functions, Glycoproteins having homogeneous oligosaccharides that can be varied as much as chemists like are requisite. Chemical synthesis of Glycoproteins recently emerged and provides homogeneous Glycoproteins such as erythropoietin. For this purpose, an efficient preparation of complex-type oligosaccharides, solid-phase glycopeptide synthesis, glycopeptide segment coupling, and glycopeptide folding procedure are essential. This chapter introduces typical procedure for the chemical synthesis of homogeneous Glycoprotein.

Masayuki Izumi – One of the best experts on this subject based on the ideXlab platform.

  • monitoring of Glycoprotein quality control system with a series of chemically synthesized homogeneous native and misfolded Glycoproteins
    Journal of the American Chemical Society, 2018
    Co-Authors: Tatsuto Kiuchi, Masayuki Izumi, Akira Seko, Masafumi Sakono, Ryo Okamoto, Yuki Mukogawa, Arisa Shimada, Yoichi Takeda, Yasuhiro Kajihara
    Abstract:

    The Glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:Glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded Glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous Glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-β (IFN-β), and interleukin 8 (IL8) and their misfolded counterparts, and used these Glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded Glycoproteins to native form through folding intermediates, allowing for the relationship between the amou…

  • chemical synthesis of homogeneous Glycoproteins for the study of Glycoprotein quality control system
    Israel Journal of Chemistry, 2015
    Co-Authors: Masayuki Izumi, Simone Dedola, Yasuhiro Kajihara
    Abstract:

    The Glycoprotein quality control system exists in the endoplasmic reticulum to maintain protein homeostasis and prevent accumulation of aberrant Glycoproteins. Folding sensor enzyme uridine diphosphate (UDP)glucose : Glycoprotein glucosyltransferase (UGGT) plays an important role in this system through its ability to discriminate immature or misfolded Glycoproteins from native ones. UGGT transfers a glucose residue to a Glycoprotein containing Man9GlcNAc2 (M9; Man=mannose, GlcNAc=N-acetyl-D-glucosamine) N-glycan only when the Glycoprotein has not attained a native form. We chemically prepared homogeneous Glycoproteins containing M9 N-glycan in the native form as well as in misfolded forms and examined them as substrates of UGGT. Glucose transfer to misfolded Glycoproteins was clearly observed by LC-MS, but Glycoproteins in the native form were barely glucosylated. Furthermore, we constructed an in vitro Glycoprotein folding system in the presence of UGGT and found out that all folding intermediates which appeared during folding were also glucosylated. Through these experiments, we demonstrated the usefulness of chemically synthesized homogeneous Glycoproteins as probes to gain insights into the molecular basis of the Glycoprotein quality control system.

  • Chemical Synthesis of Homogeneous Glycoproteins
    Glycoscience: Biology and Medicine, 2014
    Co-Authors: Masayuki Izumi, Ryo Okamoto, Yasuhiro Kajihara
    Abstract:

    Oligosaccharides of Glycoprotein are known to be heterogeneous. These diverse oligosaccharide profiles have been a hindrance to elucidating oligosaccharide functions in many biological events. In order to elucidate oligosaccharide functions, Glycoproteins having homogeneous oligosaccharides that can be varied as much as chemists like are requisite. Chemical synthesis of Glycoproteins recently emerged and provides homogeneous Glycoproteins such as erythropoietin. For this purpose, an efficient preparation of complex-type oligosaccharides, solid-phase glycopeptide synthesis, glycopeptide segment coupling, and glycopeptide folding procedure are essential. This chapter introduces typical procedure for the chemical synthesis of homogeneous Glycoprotein.

Heinz Feldmann – One of the best experts on this subject based on the ideXlab platform.

  • Intracellular localization of Crimean-Congo Hemorrhagic Fever (CCHF) virus Glycoproteins
    Virology Journal, 2005
    Co-Authors: Sebastian Haferkamp, Heinz Feldmann, Lisa Fernando, Tino F Schwarz, Ramon Flick
    Abstract:

    Background Crimean-Congo Hemorrhagic Fever virus (CCHFV), a member of the genus Nairovirus , family Bunyaviridae , is a tick-borne pathogen causing severe disease in humans. To better understand the CCHFV life cycle and explore potential intervention strategies, we studied the biosynthesis and intracellular targeting of the Glycoproteins, which are encoded by the M genome segment. Results Following determination of the complete genome sequence of the CCHFV reference strain IbAr10200, we generated expression plasmids for the individual expression of the Glycoproteins G_N and G_C, using CMV- and chicken β-actin-driven promoters. The cellular localization of recombinantly expressed CCHFV Glycoproteins was compared to authentic Glycoproteins expressed during virus infection using indirect immunofluorescence assays, subcellular fractionation/western blot assays and confocal microscopy. To further elucidate potential intracellular targeting/retention signals of the two Glycoproteins, GFP-fusion proteins containing different parts of the CCHFV Glycoprotein were analyzed for their intracellular targeting. The N-terminal Glycoprotein G_N localized to the Golgi complex, a process mediated by retention/targeting signal(s) in the cytoplasmic domain and ectodomain of this protein. In contrast, the C-terminal Glycoprotein G_C remained in the endoplasmic retireticulum but could be rescued into the Golgi complex by co-expression of G_N. Conclusion The data are consistent with the intracellular targeting of most bunyavirus Glycoproteins and support the general model for assembly and budding of bunyavirus particles in the Golgi compartment.

  • properties of replication competent vesicular stomatitis virus vectors expressing Glycoproteins of filoviruses and arenaviruses
    Journal of Virology, 2004
    Co-Authors: Michael Garbutt, Ryan Liebscher, Victoria Wahljensen, Steven M Jones, Peggy Moller, Ralf Wagner, Viktor E Volchkov, Hansdieter Klenk, Heinz Feldmann
    Abstract:

    Replication-competent recombinant vesicular stomatitis viruses (rVSVs) expressing the type I transmembrane Glycoproteins and selected soluble Glycoproteins of several viral hemohemorrhagic fever agents (Marburg virus, Ebola virus, and Lassa virus) were generated and characterized. All recombinant viruses exhibited rhabdovirus morphology and replicated cytolytically in tissue culture. Unlike the rVSVs with an additional transcription unit expressing the soluble Glycoproteins, the viruses carrying the foreign transmembrane Glycoproteins in replacement of the VSV Glycoprotein were slightly attenuated in growth. Biosynthesis and processing of the foreign Glycoproteins were authentic, and the cell tropism was defined by the transmembrane Glycoprotein. None of the rVSVs displayed pathogenic potential in animals. The rVSV expressing the Zaire Ebola virus transmembrane Glycoprotein mediated protection in mice against a lethal Zaire Ebola virus challenge. Our data suggest that the recombinant VSV can be used to study the role of the viral Glycoproteins in virus replication, immune response, and pathogenesis.

  • the hantaan virus Glycoprotein precursor is cleaved at the conserved pentapeptide waasa
    Virology, 2001
    Co-Authors: Christian Lober, Hansdieter Klenk, Barbel Anheier, Steffi Lindow, Heinz Feldmann
    Abstract:

    Abstract The medium segment of the tripartite negative-stranded RNA genome of hantaviruses encodes for the predicted Glycoprotein precursor GPC. We have demonstrated here the expression of the Glycoprotein precursor of Hantaan virus following transfection of mammalian cells. The cleavage of the precursor into the Glycoproteins G1 and G2 followed the rules for signal peptides and seemed to occur directly at the pentapeptide motif “WAASA.” Our data indicate that the signal peptidase complex is responsible for the proteolytic processing of the precursor GPC of Hantaan virus. The comparison of this region of the Glycoprotein precursor, including the absolutely conserved WAASA motif, suggests a similar cleavage event for all hantavirus Glycoproteins.

Joseph Sodroski – One of the best experts on this subject based on the ideXlab platform.

  • Subunit Stoichiometry of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers during Virus Entry into Host Cells
    Journal of Virology, 2006
    Co-Authors: Xinzhen Yang, Svetla Kurteva, Joseph Sodroski
    Abstract:

    The envelope Glycoproteins of human immunodeficiency viruvirus type 1 (HIV-1) function as a homotrimer of gp120/gp41 heterodimers to support virus entry. During the process of virus entry, an individual HIV-1 envelope Glycoprotein trimer binds the cellular receptors CD4 and CCR5/CXCR4 and mediates the fusion of the viral and the target cellular membranes. By studying the function of heterotrimers between wild-type and nonfunctional mutant envelope Glycoproteins, we found that two wild-type subunits within an envelope Glycoprotein trimer are required to support virus entry. Complementation between HIV-1 envelope Glycoprotein mutants defective in different functions to allow virus entry was not evident. These results assist our understanding of the mechanisms whereby the HIV-1 envelope Glycoproteins mediate virus entry and membrane fusion and guide attempts to inhibit these processes.

  • replicative function and neutralization sensitivity of envelope Glycoproteins from primary and t cell line passaged human immunodeficiency virus type 1 isolates
    Journal of Virology, 1995
    Co-Authors: Nancy Sullivan, J Li, Wolfgang Hofmann, Joseph Sodroski
    Abstract:

    The structure, replicative properties, and sensitivity to neutralization by soluble CD4 and monoclonal antibodies were examined for molecularly cloned envelope Glycoproteins derived from human immunodeficiency viruvirus type 1 (HIV-1) viruses either isolated directly from patients or passaged in T-cell lines. Complementation of virus entry into peripheral blood mononuclear cell targets by primary patient envelope Glycoproteins exhibited efficiencies ranging from that observed for the HXBc2 envelope Glycoproteins, which are derived from a T-cell line-passaged virus, to approximately fivefold-lower values. The ability of the envelope Glycoproteins to complement virus entry roughly correlated with sensitivity to neutralization by soluble CD4. Laboratory-adapted viruses were sensitive to neutralization by monoclonal antibodies directed against the CD4-binding site and the third variable (V3) loop of the gp120 Glycoprotein. By comparison, viruses with envelope Glycoproteins from primary patient isolates exhibited decreased sensitivity to neutralization by these monoclonal antibodies; for these viruses, neutralization sensitivity correlated with replicative ability. Subinhibitory concentrations of soluble CD4 and a CD4-binding site-directed antibody significantly enhanced the entry of viruses containing envelope Glycoproteins from some primary patient isolates. The sensitivity of viruses containing the different envelope Glycoproteins to neutralization by soluble CD4 or monoclonal antibodies could be predicted by assays dependent on the binding of the inhibitory molecule to the oligomeric envelope Glycoprotein complex but less well by assays measuring binding to the monomeric gp120 Glycoprotein. These results indicate that the intrinsic structure of the oligomeric envelope Glycoprotein complex of primary HIV-1 isolates, while often less than optimal with respect to the mediation of early events in virus replication, allows a relative degree of resistance to neutralizing antibodies. The interplay of selective forces for higher virus replication efficiency and resistance to neutralizing antibodies could explain the temporal course described for the in vivo emergence of HIV-1 isolates with differing phenotypes.

  • effect of amino acid changes in the v1 v2 region of the human immunodeficiency virus type 1 gp120 Glycoprotein on subunit association syncytium formation and recognition by a neutralizing antibody
    Journal of Virology, 1993
    Co-Authors: Nancy Sullivan, Markus Thali, C Furman, David D Ho, Joseph Sodroski
    Abstract:

    The contributions of the first and second variable regions of the human immunodeficiency viruvirus type 1 gp120 Glycoprotein to envelope Glycoprotein structure, function, and recognition by a neutralizing antibody were studied. Several mutants with substitutions in the V2 loop demonstrated complete dissociation of the gp120 and gp41 Glycoproteins, suggesting that inappropriate changes in V2 conformation can affect subunit assembly. Some Glycoproteins with changes in V1 or V2 were efficiently expressed on the cell surface and were able to bind CD4 but were deficient in syncytium formation and/or virus entry. Recognition of gp120 by the neutralizing monoclonal antiantibody G3-4 was affected by particular substitutions affecting residues 176 to 184 in the V2 loop. These results suggest that the V1/V2 variable regions of the human immunodeficiency viruvirus type 1 gp120 Glycoprotein play a role in postreceptor binding events in the membrane fusifusion process and can act as a target for neutralizing antibodies. Images

Simone Dedola – One of the best experts on this subject based on the ideXlab platform.

  • chemical synthesis of homogeneous Glycoproteins for the study of Glycoprotein quality control system
    Israel Journal of Chemistry, 2015
    Co-Authors: Masayuki Izumi, Simone Dedola, Yasuhiro Kajihara
    Abstract:

    The Glycoprotein quality control system exists in the endoplasmic reticulum to maintain protein homeostasis and prevent accumulation of aberrant Glycoproteins. Folding sensor enzyme uridine diphosphate (UDP)glucose : Glycoprotein glucosyltransferase (UGGT) plays an important role in this system through its ability to discriminate immature or misfolded Glycoproteins from native ones. UGGT transfers a glucose residue to a Glycoprotein containing Man9GlcNAc2 (M9; Man=mannose, GlcNAc=N-acetyl-D-glucosamine) N-glycan only when the Glycoprotein has not attained a native form. We chemically prepared homogeneous Glycoproteins containing M9 N-glycan in the native form as well as in misfolded forms and examined them as substrates of UGGT. Glucose transfer to misfolded Glycoproteins was clearly observed by LC-MS, but Glycoproteins in the native form were barely glucosylated. Furthermore, we constructed an in vitro Glycoprotein folding system in the presence of UGGT and found out that all folding intermediates which appeared during folding were also glucosylated. Through these experiments, we demonstrated the usefulness of chemically synthesized homogeneous Glycoproteins as probes to gain insights into the molecular basis of the Glycoprotein quality control system.

  • Folding of Synthetic Homogeneous Glycoproteins in the Presence of a Glycoprotein Folding Sensor Enzyme
    Angewandte Chemie, 2014
    Co-Authors: Simone Dedola, Masayuki Izumi, Yutaka Makimura, Akira Seko, Akiko Kanamori, Masafumi Sakono, Yukishige Ito, Yasuhiro Kajihara
    Abstract:

    UDP-glucose:Glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded Glycoproteins in the Glycoprotein quality control system of the endoplasmic retireticulum. UGGT detects misfolded Glycoproteins and re-glucosylates them as a tag for misfolded Glycoproteins. A flexible model to reproduce in vitro folding of a Glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded Glycoproteins is described. The data demonstrates that UGGT can re-glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the Glycoprotein folding intermediates.