title: Characterisation of the enzymatic complex for the first step in glycosylphosphatidylinositol biosynthesis.
abstract: The mammalian N-acetylglucosaminyl transferase for the first step in glycosylphosphatidylinositol biosynthesis has been shown to consist of at least four components: PIG-A, PIG-C, PIG-H and GPI1. Here, the enzymatic complex is further characterised. PIG-A protein, which is thought to represent the catalytic subunit of the complex, was expressed in an epitope-tagged form in the PIG-A deficient JY5 lymphoblastoid cell line. Subcellular localisation of this protein was studied using immunofluorescence and immunoelectron microscopy. The protein was localised to both perinuclear and mitochondria-associated lamellae of the endoplasmic reticulum. Using affinity chromatography, epitope-tagged PIG-A protein was partially purified. To identify regions that might be involved in the catalytic process, computer-aided comparison was performed between PIG-A and 26 distantly related glycosyl transferases. A number of residues in the membrane-proximal region of the cytoplasmic domain (230-340) were found highly conserved. Finally, a topological model of the four partners participating in the enzymatic complex is introduced to provide a working model for further structural and functional analysis.
authors.affiliation: Department of Clinical Immunology, Hannover Medical School, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Tiede
authors.forename: C
authors.initials: C
authors.lastname: Nischan
authors.forename: J
authors.initials: J
authors.lastname: Schubert
authors.forename: R E
authors.initials: RE
authors.lastname: Schmidt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009097:Multienzyme Complexes; C080086:phosphatidylinositol glycan-class A protein; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase