Biosynthesis of GPI-anchored proteins

A typical GPI-anchor structure in human erythrocytes. GalNAc means N-acetyl galactosamine. Waved lines indicate alkyl chains. [Reproduced with permission from M. Tomita, Biochemical background of paroxysmal nocturnal hemoglobinuria. Biochem. Biophys. Acta 1455, 269 (1999).] 

CHAPTER 16 Carbohydrate Metabolism III Glycoproteins, Glycolipids, GPI Anchors, Proteoglycans, and Peptidoglycans 

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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... 

Biosynthesis of GPI-anchored proteins attachment of GPIs to proteins. 53... 

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]. 

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. 

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. 

Dolichol-P-mannose is the mannose donor for all three mannosylation steps, whereas phosphatidylethanolamine contributes the phosphoethanolamine residues. Both these precursors are synthesized on the cytoplasmic face of the ER and must be flipped into the ER lumen to participate in GPI biosynthesis. The mannosyltransferases required for the assembly of the first, second, and third mannoses are PIG-M/PIG-X, PIG-V, and PIG-B (a fourth mannose residue that is essential for GPI assembly in yeast is transferred by the protein SMP3). Phosphoethanolamine side chains attached to the first, second, and third mannose residues are transferred by PIG-N, hGPI7/PIG-F, and PIG-O/PIG-F. Fully assembled GPI structures, or the GPI moiety in GPI-anchored proteins, are frequently subject to lipid re-modeling reactions in which fatty acids or the entire lipid structure is replaced with different fatty acids or lipids. 

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. 

Zhang, H. Bradley, A. (1996) Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development 122,2977-2986. Takahama, Y., Ohishi, K Tokoro, K Sugawara, T, Yoshimura, Y, Okabe, M., et al. (1997) T cell-specific disruption of Pig-a gene involved in glycosylphosphatidyli-nositol (GPl) biosynthesis Generation and function of T cells deficient in GPI-anchored proteins. Proc. Natl. Acad. Sci. USA, in press. 

In plant tissues two major forms of polyprenols are found, e.g. free alcohols and esters with fatty acids. In some species one form is highly dominating. Similarly in animal tissues dolichols and dolichyl esters were described, accompanied by usually small amounts of dolichyl phosphate. Biological role of this latter compound is well established. Dolichyl phosphate is involved as cofactor in biosynthesis of glycoproteins and GPI-anchored proteins [3]. Biological role of free alcohol and its acyl ester has not been defined yet. As the components of biological membranes these compounds modulate membrane properties. Dolichol increases fluidity and permeability and decreases the stability of model membranes [4]. Experiments with polyprenols point to the same direction polyprenols and polyprenyl phosphates increases the fluidity and permeability of model membranes [5], Both polyprenols and dolichols could also serve as a reserve isoprenoid pool utilized by corresponding kinases. 

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). 

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... 

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. 

The cellular form of the prion protein, called PrP, is synthesized as a 253 amino acid precursor protein in humans (Figure 29. IB) (Prusiner, 1991). The N-terminal 22 amino acid residues serve as the signal peptide that allows insertion of the nascent PrP peptide into the secretory pathway during biosynthesis. The C-terminal 22 amino acid residues are involved in the covalent addition of the glycosylphosphatidylino-sitol (GPI) moiety to serine at amino acid 231 (i.e., Ser 9-The GPI anchor tethers PrP to the exhacellular side of the plasma membrane. PrP has two N-hnked glycosylation sites (Asn and Asn ) that are the sites of attachment of complex... 

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]. 

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