GPI anchors biosynthesis

J. Takeda and T. Kinoshita GPI-anchor biosynthesis. Trends in Biological Science 20 367 (1995). 

Finally, GPI-anchored proteins can be metabolically radiolabeled using radiolabeled precursors for the GPI anchor biosynthesis. Lipids, myo-inositol, ethanolamine, glucosamine or mannose have all been used to incorporate a radiolabeled tag into GPI anchors [48]. 

Analysis of GPI anchor biosynthesis was greatly facilitated by the development of a cell free system in trypanosomes [62]. The convenience of this system can not be overstated. It is a highly stable system which can be stored frozen for long periods... 

The first step in the synthesis of GPI anchors is the transfer of fV-acetylglucosamine (Glc-NAc) from UDP-GlcNAc to phosphatidylinositol (PI) (Fig. 3). This step appears to be regulated by at least three genes. At present, little is known concerning the enzymes catalyzing GPI anchor biosynthesis. Nevertheless, a panel of eight complementation... 

Mannosamine inhibits GPI anchor biosynthesis [81]. Mannosamine incorporates almost exclusively into the second mannose position of the GPI [82] and prevents further elongation of the chain, thus, acting as a chain terminator [83,84]. 

The most likely candidate for the phosphoethanolamine donor is phosphatidylethanol-amine. Metabolic radiolabeling studies demonstrate that [ H]ethanolamine is incorporated into CDP-phosphoethanolamine before it is transferred to phosphatidylethanolamine and attached to the GPI core glycan. CDP-ethanolamine, however, is not a donor since it is not required for, nor does it affect, GPI anchor biosynthesis [62]. Consistent with this observation is the lack of [ HJglucosamine incorporation into GPI anchors in yeast mutants that do not synthesize phosphatidylethanolamine from CDP-ethanolamine yet can construct GPI anchors [88]. Direct evidence for a phosphatidylethanolamine donor is still lacking. 

Prior to protein attachment, the fatty acids of the GPI anchor may be replaced in a conversion process termed fatty acid remodeling [91]. Although myristate is the sole fatty acid component in mature trypanosome GPI anchors, earlier GPI intermediates contain more hydrophobic stearate fatty acids. These longer fatty acid chains are replaced by an alternating sequence of removal and replacement of a fatty acid from each position on the glycerol [91]. Lipid remodeling may not be unique to trypanosome GPI anchor biosynthesis since mature yeast GPI anchors contain ceramide, whereas an early yeast GPI intermediate has a diacylglycerol species [33]. 

Evidence that protein addition to GPI anchors occurs in the endoplasmic reticulum stems from kinetic studies on GPI anchor addition to newly synthesized proteins [97,98], the accumulation of unprocessed precursor proteins in the endoplasmic reticulum of yeast and mammalian mutants in GPI anchor biosynthesis [111,112], and the use of a microsomal assay system to analyze the C-terminal processing of GPI-linked proteins [113]. The biosynthesis of GPI anchors also is assumed to be localized to the endoplasmic reticulum. This assumption is well founded since GlcNAc-PI transferase and deacetylase activity co-fractionate with markers for the endoplasmic reticulum [114]. Therefore, at least the initial steps in GPI anchor biosynthesis occur in the endoplasmic reticulum. 

Defects in GPI anchor biosynthesis paroxysmal nocturnal hemoglobinuria... 

From this network it is clearly visible, that LPG Biosynthetic Pathway, GIPL Biosynthesis Pathway, GPI anchor Biosynthesis Pathway, and Dolichyl-diphosphooligosaccharide biosynthesis Pathway are inter-connected through one or more genes involved commonly in the regulation of the proteins which act as enzymes catalyzing the reactions in these pathways (Fig.5). 

Thereby, from the Cytoscape network and Fig 6, it is established that majority of the enzymes in a large number are present only in the LPG, GIPL and GPI anchor bioynthesis pathways, thus giving a wide scope for the enzymatic studies to be undertaken. Consequently, the Lipophosphoglycan Pathway in conjunction with the GIPL and GPI anchor biosynthesis is of paramount importance. 

E. Canivene-Gansel, I. Imhof, F. Reggiori F, P. Burda, A. Conzelmann, and A. Benachour, GPI anchor biosynthesis in yeast phosphoethanolamine is attached to the al,4-linked mannose of the complete precursor glycophospholipid, Glycobiology 1998, 8, 761 770. 

T. Miyata, J. Takeda, Y. lida, N. Yamada, N. Inoue, M. Takahashi, K. Maeda, T. Kitani T. Kinoshita. The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis. Science, 1993, 259, 1318-1320. 

K. Kawagoe, J. Takeda, Y. Endo T. Kinoshita. Molecular cloning of murine pig-a, a gene for GPI-anchor biosynthesis, and demonstration of interspecies conservation of its structure, function, and genetic locus. Genomics, 1994, 23, 566-574. 

W. J. Masterson M. A. Ferguson. Phenylmethanesulphonyl fluoride inhibits GPI anchor biosynthesis in the African trypanosome. EMBO J, 1991, 10, 2041 -2045. 

---