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Preparation of Quality Inositol Pyrophosphates
10:34

Preparation of Quality Inositol Pyrophosphates

Published on: September 3, 2011

Characterisation of the enzymatic complex for the first step in glycosylphosphatidylinositol biosynthesis

A Tiede1, C Nischan, J Schubert

  • 1Department of Clinical Immunology, Hannover Medical School, Germany.

The International Journal of Biochemistry & Cell Biology
|March 15, 2000
PubMed

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.

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