The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Howard Riezman - One of the best experts on this subject based on the ideXlab platform.

  • The presence of an ER exit signal determines the protein sorting upon ER exit in yeast
    2008
    Co-Authors: Reika Watanabe, Guillaume A. Castillon, Anja Meury, Howard Riezman
    Abstract:

    In yeast, there are at least two vesicle populations upon ER (endo-plasmic reticulum) exit, one containing Gap1p (general amino-acid permease) and a glycosylated α-factor, gpαF (glycosylated proα-factor), and the other containing GPI (glycosylphospha-tidylinositol)-anchored proteins, Gas1p (glycophospholipid-anchored surface protein) and Yps1p. We attempted to identify sorting determinants for this protein sorting event in the ER. We found that mutant Gas1 proteins that lack a GPI anchor and/ or S/T region (serine- and threonine-rich region), two common characteristic features conserved among yeast GPI-anchored proteins, were still sorted away from Gap1p-containing vesicles. Furthermore, a mutant glycosylated α-factor, gpαGPI, which contains both the GPI anchor and S/T region from Gas1p, still entered Gap1p-containing vesicles, demonstrating that these conserved characteristics do not prevent proteins from enterin

  • determinants for glycophospholipid anchoring of the saccharomyces cerevisiae gas1 protein to the plasma membrane
    Molecular and Cellular Biology, 1991
    Co-Authors: C Nuoffer, P Jeno, Andreas Conzelmann, Howard Riezman
    Abstract:

    A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid. We have cloned the gene (GAS1) encoding the 125-kDa protein (Gas1p) and found that the function of Gas1p is not essential for cell viability. The nucleotide sequence of GAS1 predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N- and O-linked glycosylation, and a C-terminal hydrophobic domain. Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gas1p is removed during anchor addition. However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane. Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence. We conclude that glycophospholipid anchoring of Gas1p depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.

Ingrid W Caras - One of the best experts on this subject based on the ideXlab platform.

  • An internally positioned signal can direct attachment of a glycophospholipid membrane anchor.
    The Journal of cell biology, 1991
    Co-Authors: Ingrid W Caras
    Abstract:

    All known glycophosphatidylinositol (GPI)-anchored membrane proteins contain a COOH-terminal hydrophobic domain necessary for signalling anchor attachment. To examine the requirement that this signal be at the COOH terminus of the protein, we constructed a chimeric protein, DAFhGH, in which human growth hormone (hGH) was fused to the COOH terminus of decay accelerating factor (DAF) (a GPI-anchored protein), thereby placing the GPI signal in the middle of the chimeric protein. We show that the fusion protein appears to be processed at the normal DAF processing site in COS cells, producing GPI-anchored DAF on the cell surface. This result indicates that the GPI signal does not have to be at the COOH terminus to direct anchor addition, suggesting that the absence of a hydrophilic COOH-terminal extension (beyond the hydrophobic domain) is not a necessary requirement for GPI anchoring. A similar DAFhGH fusion, containing an internal GPI signal in which the DAF hydrophobic domain was replaced with the signal peptide of hGH, also produced GPI-anchored cell surface DAF. The signal for GPI attachment thus exhibits neither position specificity nor sequence specificity. In addition, mutant DAF or DAFhGH constructs lacking an NH2-terminal signal peptide failed to produce GPI-anchored protein, suggesting that membrane translocation is necessary for anchor addition.

  • glycophospholipid membrane anchor attachment molecular analysis of the cleavage attachment site
    Journal of Biological Chemistry, 1991
    Co-Authors: Paul Moran, Helga Raab, William J Kohr, Ingrid W Caras
    Abstract:

    The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophosphatidylinositol (GPI) membrane anchor in a process involving proteolytic removal of 17-31 COOH-terminal residues. Previous work suggested that two elements are required for anchor addition, a COOH-terminal hydrophobic domain (the GPI signal) and an element located NH2-terminal to it, postulated to be the cleavage/attachment site. Using [3H]ethanolamine (a component of the anchor) to tag the COOH terminus, we isolated and sequenced a COOH-terminal tryptic peptide, thereby identifying Ser-319 as the COOH-terminal residue attached to the GPI anchor. This indicates that a 28-residue peptide is removed during processing and localizes the cleavage/attachment site precisely to the region previously shown to be required for anchor attachment (between 10 and 20 residues NH2-terminal to the hydrophobic domain). Since DAF contains multiple cryptic cleavage/attachment sites, we used a GPI-linked human growth hormone-DAF fusion to study the structural requirements for cleavage/attachment. Our results show that while sequences immediately NH2-terminal to the attachment site are not required for anchor addition, deletion of Ser-319 abolishes both anchor attachment and transport to the cell surface. Systematic replacement of the attachment site serine with all possible amino acids indicated that alanine, aspartate, asparagine, glycine, or serine efficiently support GPI anchor attachment while valine and glutamate are partially effective. All other substitutions including cysteine (permitted at the attachment site in other GPI-anchored proteins) abolish both GPI anchor attachment and transport to the cell surface, resulting in accumulation of uncleaved fusion protein in internal compartments (endoplasmic reticulum and Golgi). These results support the general rule that the residue at the cleavage/attachment site must be small. Further, addition of a GPI anchor appears to be necessary for transport to the cell surface in transfected COS cells.

Takao Taki - One of the best experts on this subject based on the ideXlab platform.

  • structure of a novel phosphocholine containing aminoglycoglycerolipid of mycoplasma fermentans
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Kazuhiro Matsuda, Ineo Ishizuka, Takeshi Kasama, Naoki Yamamoto, Shizuo Handa, Takao Taki
    Abstract:

    Abstract Mycoplasma fermentans is thought to be a pathogen of rheumatoid arthritis or a cofactor of AIDS (acquired immunodeficiency syndrome). To elucidate the possible involvement of membrane constituents in the pathogenesis of these diseases, we studied its lipid components. Several alkali labile Glycophospholipids were detected and named glyco-glycerophospholipids (GGPLs). Previously, we purified and determined the structure of one of them as 6′-O-phosphocholine-α-glucopyranosyl-(1′-3)-1,2-diacyl-sn-glycerol (GGPL-I). The present paper describes the purification and structural characterization of GGPL-III, the major GGPL of M. fermentans using 1H-, 13C- and 31P-nuclear magnetic resonance spectroscopy, and mass-spectroscopy as 1″-phosphocholine,2″-amino dihydroxypropane-3″-phospho-6′-α-glucopyranosyl-(1′-3)-1,2-diacyl-glycerol.

  • structure of a novel phosphocholine containing glycoglycerolipid from mycoplasma fermentans
    Journal of Biological Chemistry, 1994
    Co-Authors: Kazuhiro Matsuda, Ineo Ishizuka, Takeshi Kasama, Naoki Yamamoto, Shizuo Handa, Takao Taki
    Abstract:

    Abstract Mycoplasma fermentans is thought to be a pathogen of rheumatoid arthritis or cofactor of AIDS. A novel phosphocholine-containing glycoglycerophospholipid named GGPL-I was isolated from a M. fermentans-infected human helper T-cell culture. It was revealed that GGPL-I is a lipid component of the M. fermentans and a major immunological determinant. The GGPL-I was purified by DEAE-Sephadex column chromatography and repeated Iatrobeads column chromatography. The purified glycophospholipid was subjected to structural characterization by thin-layer chromatography, Fourier-transform infrared spectrometry, liquid secondary ion mass spectrometry, and nuclear magnetic resonance spectroscopy. Its structure was determined to be as follows: 6'-O-phosphocholine-alpha-glucopyranosyl-(1'-3)-1,2-diacyl-sn-glycerol. This glycoglycerophospholipid is unique in containing phosphocholine, which is attached to C-6 of glucose. The stereospecific numbering (sn) of naturally occurring GGPL-I was determined through comparison with chemically synthesized compounds.

Spencer J. Williams - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis of glycoconjugate fragments of mycobacterial phosphatidylinositol mannosides and lipomannan
    Beilstein journal of organic chemistry, 2011
    Co-Authors: Benjamin Cao, Jonathan M. White, Spencer J. Williams
    Abstract:

    Mycobacterium tuberculosis, the causitive agent of tuberculosis (TB), possesses a complex cell wall containing mannose-rich glycophospholids termed phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). These Glycophospholipids play important roles in cell wall function and host–pathogen interactions. Synthetic PIM/LM/LAM substructures are useful biochemical tools to delineate and dissect the fine details of mannose glycophospholipid biosynthesis and their interactions with host cells. We report the efficient synthesis of a series of azidooctyl di- and trimannosides possessing the following glycan structures: α-Man-1,6-α-Man, α-Man-1,6-α-Man-1,6-α-Man, α-Man-1,2-α-Man-1,6-α-Man and 2,6-di-(α-Man)-α-Man. The synthesis includes the use of non-benzyl protecting groups compatible with the azido group and preparation of the branched trisaccharide structure 2,6-di-(α-Man)-α-Man through a double glycosylation of a 3,4-butanediacetal-protected mannoside. The azidooctyl groups of these synthetic mannans were elaborated to fluorescent glycoconjugates and squaric ester derivatives useful for further conjugation studies.

  • chemical approaches for the study of the mycobacterial glycolipids phosphatidylinositol mannosides lipomannan and lipoarabinomannan
    Natural Product Reports, 2010
    Co-Authors: Spencer J. Williams
    Abstract:

    Covering: up to October 2009 The mannose-rich mycobacterial Glycophospholipids phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM) are important constituents of the mycobacterial cell wall and are critical mediators of host–pathogen interactions. Since the earliest reports of their existence almost 80 years ago, intensive efforts have now resulted in an almost complete description of their molecular structures. In parallel, based on studies over the last 10 years, their biosynthesis is now reasonably well understood. Owing to the structural complexity of these Glycophospholipids, chemically synthesized fragments have been essential for dissecting their biosynthesis and biological functions. This review provides an overview of the synthesis of fragments of the PIMs, LM and LAM, and approaches to the total synthesis of PIMs and analogues. Also covered are uses of synthetic fragments, authentic structures and analogues as biochemical reagents and immunomodulators.

Eric Hunter - One of the best experts on this subject based on the ideXlab platform.

  • expression and characterization of glycophospholipid anchored human immunodeficiency virus type 1 envelope glycoproteins
    Journal of Virology, 1993
    Co-Authors: Karl Salzwedel, Patrick B Johnston, S J Roberts, John W Dubay, Eric Hunter
    Abstract:

    Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome.