Glycosylphosphatidylinositol Transamidase Research Articles

Gaa1p and gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins.

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted a GAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.

Pig-n, a Mammalian Homologue of Yeast Mcd4p, Is Involved in Transferring Phosphoethanolamine to the First Mannose of the Glycosylphosphatidylinositol

Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins. The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains. We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p. We disrupted the gene in F9 embryonal carcinoma cells. In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine. Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine. The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells. An inhibitor of GPI biosynthesis, YW3548/BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins. Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug.

GAA1 recognizes the GPI anchor attachment signals and is complexed with GP18 to form a GPI transamidase

Crude extract was prepared from the sea anemone, Bunodosoma cavernata. The protein content of the extract was estimated to be 0.52 mg protein/ml. The extract was standardized based on the percentage inhibition of histamine-induced contraction of the guinea pig ileum, to determine the biological unit of activity (AU) of the extract. As extracts prepared on different occasions lost potency on storage, the stability of the extract was also investigated. Extracts prepared from fresh animals were about 15% more potent than those prepared from freeze-dried animals. However, freeze-dried animal extracts maintained their potency for about 6 months under storage at −20°C. Lethality studies gave an ld50 of 40 μg protein/kg mice i.p. Also, the crude extract dose-dependently hemolyzed human erythrocytes at room temperature. This activity was favoured by higher temperatures, which peaked at about 60°C, and by pH in the alkaline range. We conclude that the crude extract from B. cavernata, though highly toxic, may also contain some biologically active agents which include a haemolytic factor and antihistamine(s), as indicated by its histamine-blocking action.

The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase.

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5' extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans approximately 25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.

Mutational analysis of the C-terminal signal peptide of bovine liver 5′-nucleotidase for GPI anchoring: a study on the significance of the hydrophilic spacer region

Bovine liver 5′-nucleotidase is a GPI-anchored protein whose Ser 523 attaches to GPI as the ω-site. For GPI-modification, pro-protein of the enzyme possesses a signal peptide at the C-terminus, comprising a hydrophilic spacer sequence of 8 amino acid residues and the following hydrophobic region of 17 amino acid residues. The C-terminal signal peptide is replaced by GPI on a luminal leaflet of endoplasmic reticulum. To characterize the C-terminal signal peptide for GPI modification, we constructed a series of deletion and elongation mutant genes, altering length of the hydrophilic spacer sequence by site-directed mutagenesis. Systematic deletion and Ala insertion of the sequence showed that the sequence of 6–14 residues were compatible for GPI modification. For GPI transfer to the pro-protein, the optimum length of spacer sequence would be 8, being consistent with natural selection. The spacer sequence may play a role for leading the ω-residue correctly to the active site of putative GPI transamidase. The elongation of the spacer is more permissible than deletion. Nevertheless, the length of the spacer sequence may influence efficiency of GPI modification by its positive or negative control.

A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.

Evidence that the putative COOH-terminal signal transamidase involved in glycosylphosphatidylinositol protein synthesis is present in the endoplasmic reticulum.

Nascent proteins destined to be processed to a glycosylphosphatidylinositol (GPI)-anchored membrane form contain NH2-terminal and COOH-terminal signal peptides. The first directs a nascent protein into the endoplasmic reticulum; the second peptide targets the protein to a putative COOH-terminal signal transamidase where cleavage of the peptide and addition of the GPI anchor occur. We recently showed that ATP hydrolysis is required for maturation of GPI proteins at a stage prior to transamidation. Here we show that one of the ATP-requiring proteins involved in processing of GPI-anchored proteins in the endoplasmic reticulum is the immunoglobulin heavy chain binding protein (BiP; GRP 78). This and related findings indicate that GPI transamidase is localized in the endoplasmic reticulum.

Hepatitis A virus infection among homosexual men.

<h3>Abstract</h3> <h3>Background</h3> Glycosylphosphatidylinositol (GPI) addition is one of the several post-translational modifications to proteins that increase their affinity for membranes. In eukaryotes, the GPI transamidase complex (GPI-T) catalyzes the attachment of pre-assembled GPI anchors to GPI-anchored proteins (GAPs) through a transamidation reaction. A mutation in <i>AtGPI8</i> (<i>gpi8-2</i>), the putative catalytic subunit of GPI-T in Arabidopsis, is transmitted normally through the female gametophyte (FG), indicating the FG tolerates loss of GPI transamidation. In contrast, <i>gpi8-2</i> almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that <i>gpi8-2</i> FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized. <h3>Results</h3> Here we investigated the effect of loss of <i>AtPIG-S,</i> another GPI-T subunit, in both gametophytes. Like <i>gpi8-</i>2, we showed that a mutation in <i>AtPIG-S</i> (<i>pigs-1</i>) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG, yet is transmitted normally through the FG. Conversely, <i>pigs-1</i> severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A <i>pPIGS:PIGS-GFP</i> transgene complemented these MG defects and enabled generation of <i>pigs-1/pigs-1</i> seedlings, but seemingly failed to rescue the function of AtPIG-S in the sporophyte, as <i>pigs-1/pigs-1, pPIGS:PIGS-GFP</i> seedlings died soon after germination. <h3>Conclusions</h3> Characterization of <i>pigs-1</i> provided further evidence that the FG tolerates loss of GPI transamidation more than the MG and that the MG compared to the FG may be a better haploid system to study the role of GPI-anchoring. <i>pigs-1</i> pollen develops normally and thus represent a tool in which GPI anchor biosynthesis and transamidation of GAPs have been uncoupled, offering a potential way to study free GPI in plant development. While previously reported male fertility defects of GPI biosynthesis mutants could have been due either to loss of GPI or GAPs lacking the GPI anchor, our results clarified that the loss of mature GAPs underlie male fertility defects of GPI-deficient pollen grains, as <i>pigs-1</i> is defective only in the downstream transamidation step. Our study also provided further evidence that GPI transamidation is essential in seedling development.