Transient N-acetylgalactosaminylation of mannosyl phosphate side chain in Paramecium primaurelia glycosylphosphatidylinositols.
The surface antigens of the free-living protozoan Paramecium primaurelia belong to the family of glycosylphosphatidylinositol (GPtdIns)-anchored proteins. Using a cell-free system prepared from P. primaurelia, we have described the structure and biosynthetic pathway for GPtdIns glycolipids. The core glycans of the polar glycolipids are modified by a mannosyl phosphate side chain. The data suggest that the mannosyl phosphate side chain is added onto the core glycan in two steps. The first step involves the phosphorylation of the GPtdIns trimannosyl conserved core glycan via an ATP-dependent kinase, prior to the addition of the mannose linked to the phosphate group. We show that dolichol phosphate mannose is the donor of all mannose residues including the mannose linked to phosphate. Furthermore, we were able to identify in vitro a hydrophilic intermediate containing an additional N-acetylgalactosamine linked to the mannosyl phosphate side chain. The addition of this purified hydrophilic radiolabelled intermediate into the cell-free system leads to a loss of the GalNAc residue and its conversion to the penultimate intermediate having only mannosyl phosphate as a side chain. Together the data indicate that the GalNAc-containing intermediate is a transitional intermediate. We suggest that the GalNAc-containing intermediate is essential for biosynthesis and maturation of GPtdIns precursors. It is hypothesized that this oligosaccharide processing in the course of GPtdIns biosynthesis is required for the translocation of GPtdIns from the cytoplasmic side of the endoplasmic reticulum to the luminal side.
Azzouz
N
N
Med. Zentrum für Hygiene und Medizinische Mikrobiologie, Philipps-Universität Marburg, Germany. azzouz@mailer.uni-marburg.de
Gerold
P
P
Schmidt
J
J
Capdeville
Y
Y
Schwarz
R T
RT
D000116:Acetylgalactosamine / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002240:Carbohydrate Sequence; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism; D004286:Dolichols; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism; D008969:Molecular Sequence Data; D010247:Paramecium / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism
D004286:Dolichols; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008360:Mannosephosphates; D008563:Membrane Lipids; D011134:Polysaccharides; D004287:Dolichol Monophosphate Mannose; D000255:Adenosine Triphosphate; D000116:Acetylgalactosamine