The Biosynthesis of GPI Anchors

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. 

T. Kinoshita Enzymes required for biosynthesis of GPI-anchored proteins H. Kunz Synthetic glycopeptides for the development of antitumor vaccines M. A. J. Ferguson GPI and glycoprotein biosynthesis as targets for anti-parasite design... 

An unusual structural feature from the inositol perspective is the derivatiza-tion of the 2-hydroxyl group of inositol by a long chain fatty acid (palmitic acid). In many mature GPI-proteins, the palmatoyl side chain is not present the palmatoyl chain is added during the biosynthesis of GPI and removed after the GPI anchor is attached to the protein during posttranslational modification. Although the wyo-isomer of inositol is the most prevalent form in GPI molecules, the presence of chiro-inositol has also been detected. 

Biosynthesis of GPI anchors starts with the core structure assembly by sequential addition of UDP-GlcNAc (followed by iV-deacetylation), dolichol-phosphate-mannose, and phospho-ethanolamine to phosphatidylinositol and culminates in the en bloc transfer to protein shortly after the protein is synthesized. However, the biosynthetic pathways can differ strikingly between different organisms with respect to specific modifications and fatty acid remodeling occurring after completion of the core glycan. This also applies for the point when certain modifications are introduced, e. g. before or after the transfer of the GPI-moiety to the protein. GPI anchors can be cleaved at defined positions by an array of enzymatic and chemical methods, respectively (O Fig. 5). Thus, it becomes possible to identify GPI-anchored proteins and, moreover, analyze the structure and biosynthesis of GPI anchors [103]. 

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... 

Figure 2. Schematic representation of GPI biosynthetic pathway in Trypanosoma sp. After biosynthesis of GPI anchor it is galactosylated and the VSG/procyclins polypeptide is subsequently added.

It is obvious that the biosynthesis of such a complex structure involves many proteins and steps. In fact, the anchor is synthesized in the ER, requiring a membrane-bound multistep pathway in which more than 20 gene products, mainly polytopic membrane proteins, take part." " The first two steps of the biosynthesis occur on the cytoplasmic site of the ER, and after flipping to the lumen of the ER the biosynthesis is completed. The GPI... 

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... 

Addition of phosphoethanolamine to the third mannose is the final step in the biosynthesis of common core structure of trypanosomal GPI anchors [62]. In mammalian GPI anchors, however, all three mannose residues may contain a phosphoethanolamine [85-87]. Although the sequence of mannose and phosphoethanolamine incorporation remains unclear in mammalian cells, there appears to be a ladder arrangement between two parallel pathways (Fig. 4). One rail of the ladder proceeds... 

GPI-deficient mammalian cells are viable in tissue culture and many GPI-deficient mutant cell lines have been established. However, GPI deficiency has major consequences at the level of tissues and the whole body. This was revealed in transgenic mouse models in which the PIG-A gene (required for the first step of GPI biosynthesis) was knocked out in specific tissues or in the whole animal. For example, keratinocyte-specific disruption of PIG-A caused abnormal development of skin leading to death of the mutant mice a few days after birth (M. Tarutani, 1997), and disruption of PIG-A in the whole animal resulted in embryos that did not develop beyond day 9 of gestation (M. Nozaki, 1999). A somatic mutation of PIG-A in multipotent hematopoietic human stem cells causes paroxysmal nocturnal hemoglobinuria, an acquired hemolytic disease in humans characterized by abnormal activation of complement on erythrocytes due to a deficiency of GPI-anchored complement regulatory proteins such as decay accelerating factor (N. Inoue, 2003). This disease is characterized by intravascular hemolysis and anemia. 

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. 

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. 

So far GPI biosynthesis raises many questions as its correct regulation is crucial for viability in yeast and for embryonic development in mammals. Many steps remain unclear, also due to the fact that most of the proteins involved still have to be characterized. The same is true for the biological function of the GPI anchor apart from membrane insertion. Several suggestions have been made according to which GPI anchors are targeting lipid rafts, specific intracellular compartments, or the apical membrane of polarized epithelial cells. 

---