Truncation of the caspase-related subunit (Gpi8p) of Saccharomyces cerevisiae GPI transamidase: dimerization revealed

Eukaryotic proteins can be post-translationally modified with a glycosylphosphatidylinositol (GPI) membrane anchor. This modification reaction is catalyzed by GPI transamidase (GPI-T), a multimeric, membrane-bound enzyme. Gpi8p, an essential component of GPI-T, shares low sequence similarity with caspases and contains all or part of the enzyme's active site [U. Meyer, M. Benghezal, I. Imhof, A. Conzelmann, Biochemistry 39 (2000) 3461-3471]. Structural predictions suggest that the soluble portion of Gpi8p is divided into two domains: a caspase-like domain that contains the active site machinery and a second, smaller domain of unknown function. Based on these predictions, we evaluated a soluble truncation of Gpi8p (Gpi8(23-306)). Dimerization was investigated due to the known proclivity of caspases to homodimerize; a Gpi8(23-306) homodimer was detected by native gel and confirmed by mass spectrometry and N-terminal sequencing. Mutations at the putative caspase-like dimerization interface disrupted dimer formation. When combined, these results demonstrate an organizational similarity between Gpi8p and caspases.     

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei

Many eukaryotic cell-surface proteins are post-translationally modified by a glycosylphosphatidylinositol (GPI) moiety that anchors them to the cell membrane. The biosynthesis of GPI anchors is initiated in the endoplasmic reticulum by transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol. This reaction is catalyzed by GPI GlcNAc transferase, a multisubunit complex comprising the catalytic subunit Gpi3/PIG-A as well as at least five other subunits, including the hydrophobic protein Gpi2, which is essential for the activity of the complex in yeast and mammals, but the function of which is not known. To investigate the role of Gpi2, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote and important model organism that initially provided the first insights into GPI structure and biosynthesis. We generated insect-stage (procyclic) trypanosomes that lack TbGPI2 and found that in TbGPI2-null parasites, (i) GPI GlcNAc transferase activity is reduced, but not lost, in contrast with yeast and human cells, (ii) the GPI GlcNAc transferase complex persists, but its architecture is affected, with loss of at least the TbGPI1 subunit, and (iii) the GPI anchors of procyclins, the major surface proteins, are underglycosylated when compared with their WT counterparts, indicating the importance of TbGPI2 for reactions that occur in the Golgi apparatus. Immunofluorescence microscopy localized TbGPI2 not only to the endoplasmic reticulum but also to the Golgi apparatus, suggesting that in addition to its expected function as a subunit of the GPI GlcNAc transferase complex, TbGPI2 may have an enigmatic noncanonical role in Golgi-localized GPI anchor modification in trypanosomes.     

TbGPI16 is an essential component of GPI transamidase in Trypanosoma brucei

Glycosylphosphatidylinositol (GPI) is widely used by eukaryotic cell surface proteins for membrane attachment. De novo synthesized GPI precursors are attached to proteins post-translationally by the enzyme complex, GPI transamidase. TbGPI16, a component of the trypanosome transamidase, shares similarity with human PIG-T. Here, we show that TbGPI16 is the orthologue of PIG-T and an essential component of GPI transamidase by creating a TbGPI16 knockout. TbGPI16 forms a disulfide-linked complex with TbGPI8. A cysteine to serine mutant of TbGPI16 was unable to fully restore the surface expression of GPI-anchored proteins upon transfection into the knockout cells, indicating that its disulfide linkage with TbGPI8 is important for the full transamidase activity.     

A conserved proline in the last transmembrane segment of Gaa1 is required for glycosylphosphatidylinositol (GPI) recognition by GPI transamidase

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT). Human GPIT is a multisubunit membrane-bound protein complex consisting of Gaa1, Gpi8, phosphatidylinositol glycan (PIG)-S, PIG-T, and PIG-U. The enzyme recognizes a C-terminal signal sequence in the proprotein and replaces it with a preformed GPI lipid. The nature of the functional interaction of the GPIT subunits with each other and with the proprotein and GPI substrates is largely unknown. We recently analyzed the GPIT subunit Gaa1, a polytopic protein with seven transmembrane (TM) spans, to identify sequence determinants in the protein that are required for its interaction with other subunits and for function (Vainauskas, S., Maeda, Y., Kurniawan, H., Kinoshita, T., and Menon, A. K. (2002) J. Biol. Chem. 277, 30535-30542). We showed that elimination of the C-terminal TM segment of Gaa1 allows the protein to interact with Gpi8, PIG-S, and PIG-T but renders the resulting GPIT complex nonfunctional. We now show that GPIT complexes containing C-terminally truncated Gaa1 possess a full complement of subunits and are able to interact with a proprotein substrate but cannot co-immunoprecipitate GPI. We go on to show that mutation of a conserved proline residue centrally located within the C-terminal TM span of Gaa1 is sufficient to abrogate the ability of the resulting GPIT complex to co-immunoprecipitate GPI. We suggest that the putative dynamic hinge created by the proline residue provides a structural basis for the interaction of GPI with GPIT.     

Proteomic identification of glycosylphosphatidylinositol anchor-dependent membrane proteins elevated in breast carcinoma

The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.     

Ethanolamine phosphate linked to the first mannose residue of glycosylphosphatidylinositol (GPI) lipids is a major feature of the GPI structure that is recognized by human GPI transamidase

Glycosylphosphatidylinositol (GPI) anchoring of proteins is catalyzed by GPI transamidase (GPIT), a multisubunit, endoplasmic reticulum (ER)-localized enzyme. GPIT recognizes ER-translocated proteins that have a GPI-directing C-terminal signal sequence and replaces this sequence with a preassembled GPI anchor. Although the GPI signal sequence has been extensively characterized, little is known about the structural features of the GPI lipid substrate that enable its recognition by GPIT. In a previous study we showed that mature GPIs could be co-immunoprecipitated with GPIT complexes containing functional subunits (Vainauskas, S., and Menon, A. K. (2004) J. Biol. Chem. 279, 6540-6545). We now use this approach, as well as a method that reconstitutes the interaction between GPIs and GPIT, to define the basis of the interaction between GPI and human GPIT. We report that (i) human GPIT can interact with GPI biosynthetic intermediates, not just mature GPIs competent for transfer to protein, (ii) the ethanolamine phosphate group on the third mannose residue of the GPI glycan is not critical for GPI recognition by GPIT, (iii) the ethanolamine phosphate residue linked to the first mannose of the GPI structure is a major feature of GPIs that is recognized by human GPIT, and (iv) the simplest GPI recognized by human GPIT is EtN-P-2Manalpha1-4GlcN-(acyl)-phosphatidyl-inositol. These studies define the molecular characteristics of GPI that are recognized by GPIT and open the way to identifying GPIT subunits that are involved in this process.     

Defining the boundaries of species specificity for the Saccharomyces cerevisiae glycosylphosphatidylinositol transamidase using a quantitative in?vivo assay

In eukaryotes, GPI (glycosylphosphatidylinositol) lipid anchoring of proteins is an abundant post-translational modification. The attachment of the GPI anchor is mediated by GPI-T (GPI transamidase), a multimeric, membrane-bound enzyme located in the ER (endoplasmic reticulum). Upon modification, GPI-anchored proteins enter the secretory pathway and ultimately become tethered to the cell surface by association with the plasma membrane and, in yeast, by covalent attachment to the outer glucan layer. This work demonstrates a novel in vivo assay for GPI-T. Saccharomyces cerevisiae INV (invertase), a soluble secreted protein, was converted into a substrate for GPI-T by appending the C-terminal 21 amino acid GPI-T signal sequence from the S. cerevisiae Yapsin 2 [Mkc7p (Y21)] on to the C-terminus of INV. Using a colorimetric assay and biochemical partitioning, extracellular presentation of GPI-anchored INV was shown. Two human GPI-T signal sequences were also tested and each showed diminished extracellular INV activity, consistent with lower levels of GPI anchoring and species specificity. Human/fungal chimaeric signal sequences identified a small region of five amino acids that was predominantly responsible for this species specificity.     

Function of a membrane-embedded domain evolutionarily multiplied in the GPI lipid anchor pathway proteins PIG-B,PIG-M,PIG-U,PIG-W,PIG-V,and PIG-Z

Distant homology relationships among proteins with many transmembrane regions (TMs) are difficult to detect as they are clouded by the TMs’ hydrophobic compositional bias and mutational divergence in connecting loops. In the case of several GPI lipid anchor biosynthesis pathway components, the hidden evolutionary signal can be revealed with dissectHMMER, a sequence similarity search tool focusing on fold-critical, high complexity sequence segments. We find that a sequence module with 10 TMs in PIG-W, described as acyl transferase, is homologous to PIG-U, a transamidase subunit without characterized molecular function, and to mannosyltransferases PIG-B, PIG-M, PIG-V and PIG-Z. We conclude that this new, membrane-embedded domain named BindGPILA functions as the unit for recognizing, binding and stabilizing the GPI lipid anchor in a modification-competent form as this appears the only functional aspect shared among all proteins. Thus, PIG-U's likely molecular function is shuttling/presenting the anchor in a productive conformation to the transamidase complex.     

GPI7 is the second partner of PIG-F and involved in modification of glycosylphosphatidylinositol

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). GPI is synthesized from phosphatidylinositol by stepwise reactions and attached en bloc to nascent proteins. In mammalian cells, the major GPI species transferred to proteins is termed H7. By attachment of an additional ethanolamine phosphate (EtNP) to the second mannose, H7 can be converted to H8, which acts as a minor type of protein-linked GPI and also exists as a free GPI on the cell surface. Yeast GPI7 is involved in the transfer of EtNP to the second mannose, but the corresponding mammalian enzyme has not yet been clarified. Here, we report that the human homolog of Gpi7p (hGPI7) forms a protein complex with PIG-F and is involved in the H7-to-H8 conversion. We knocked down hGPI7 by RNA interference and found that H7 accumulated with little production of H8. Immunoprecipitation experiments revealed that hGPI7 was associated with and stabilized by PIG-F, which is known to bind to and stabilize PIG-O, a protein homologous to hGPI7. PIG-O is a transferase that adds EtNP to the third mannose, rendering GPI capable of attaching to proteins. We further found that the overexpression of hGPI7 decreased the level of PIG-O and, therefore, decreased the level of EtNP transferred to the third mannose. Finally, we propose a mechanism for the regulation of GPI biosynthesis through competition between the two independent enzymes, PIG-O and hGPI7, for the common stabilizer, PIG-F.     

PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI. During this transamidation reaction, the GPI transamidase forms a carbonyl intermediate with a substrate protein. It was known that the GPI transamidase is a complex containing GAA1 and GPI8. Here, we report two new components of this enzyme: PIG-S and PIG-T. To determine roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates. We also demonstrate that PIG-S and PIG-T form a protein complex with GAA1 and GPI8, and that PIG-T maintains the complex by stabilizing the expression of GAA1 and GPI8. Saccharomyces cerevisiae Gpi16p (YHR188C) and Gpi17p (YDR434W) are orthologues of PIG-T and PIG-S, respectively.     

The β-1,6-glucan containing side-chain of cell wall proteins of Saccharomyces cerevisiae is bound to the glycan core of the GPI moiety

Abstract Cell wall proteins of Saccharomyces cerevisiae are anchored by means of a β-1,6-glucan-containing side-chain. It is not known whether this chain is linked to the protein part (e.g. through carbohydrate side-chains) or to the glycosylphosphatidylinositol (GPI) moiety of cell wall proteins. An IgA protease recognition site was introduced in Cwp2p, a β-1,6-glucosylated cell wall protein, immediately N-terminal from the omega amino acid (the attachment site of the GPI moiety). Proteolytic cleavage of this site revealed that the β-1,6-glucan epitope was not linked to the protein part. We conclude that neither N - or O -glycosylation is involved in β-glucosylation of cell wall proteins. This confirms that the glycan core of the GPI moiety is the probable β-1,6-glucan attachment site.     

Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins

Many eukaryotic proteins are anchored to the cell surface via glycosylphosphatidylinositol (GPI), which is posttranslationally attached to the carboxyl-terminus by GPI transamidase. The mammalian GPI transamidase is a complex of at least four subunits, GPI8, GAA1, PIG-S, and PIG-T. Here, we report Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the gene responsible, termed PIG-U, that encoded a 435-amino-acid hydrophobic protein. The GPI transamidase complex affinity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U and four other known components. Cells lacking PIG-U formed complexes of the four other components normally but had no ability to cleave the GPI attachment signal peptide. Saccharomyces cerevisiae Cdc91p, with 28% amino acid identity to PIG-U, partially restored GPI-anchored proteins on the surface of class U cells. PIG-U and Cdc91p have a functionally important short region with similarity to a region conserved in long-chain fatty acid elongases. Taken together, PIG-U and the yeast orthologue Cdc91p are the fifth component of GPI transamidase that may be involved in the recognition of either the GPI attachment signal or the lipid portion of GPI.     

Comparative efficiencies of C‐terminal signals of native glycophosphatidylinositol (GPI)‐anchored proproteins in conferring GPI‐anchoring

Every protein fated to receive the glycophosphatidylinositol (GPI) anchor post‐translational modification has a C‐terminal GPI‐anchor attachment signal sequence. This signal peptide varies with respect to length, content, and hydrophobicity. With the exception of predictions based on an upstream amino acid triplet termed ω→ω + 2 which designates the site of GPI uptake, there is no information on how the efficiencies of different native signal sequences compare in the transamidation reaction that catalyzes the substitution of the GPI anchor for the C‐terminal peptide. In this study we utilized the placental alkaline phosphatase (PLAP) minigene, miniPLAP, and replaced its native 3′ end‐sequence encoding ω‐2 to the C‐terminus with the corresponding C‐terminal sequences of nine other human GPI‐anchored proteins. The resulting chimeras then were fed into an in vitro processing microsomal system where the cleavages leading to mature product from the nascent preproprotein could be followed by resolution on an SDS–PAGE system after immunoprecipitation. The results showed that the native signal of each protein differed markedly with respect to transamidation efficiency, with the signals of three proteins out‐performing the others in GPI‐anchor addition and those of two proteins being poorer substrates for the GPI transamidase. The data additionally indicated that the hierarchical order of efficiency of transamidation did not depend solely on the combination of permissible residues at ω→ω + 2. J. Cell. Biochem. 84: 68–83, 2002. © 2001 Wiley‐Liss, Inc.     

Two subunits of glycosylphosphatidylinositol transamidase,GPI8 and PIG-T,form a functionally important intermolecular disulfide bridge

Many eukaryotic proteins are tethered to the plasma membrane via glycosylphosphatidylinositol (GPI). GPI transamidase is localized in the endoplasmic reticulum and mediates post-translational transfer of preformed GPI to proteins bearing a carboxyl-terminal GPI attachment signal. Mammalian GPI transamidase is a multimeric complex consisting of at least five subunits. Here we report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T, form a functionally important disulfide bond between conserved cysteine residues. GPI8 and PIG-T mutants in which relevant cysteines were replaced with serines were unable to fully restore the surface expression of GPI-anchored proteins upon transfection into their respective mutant cells. Microsomal membranes of these transfectants had markedly decreased activities in an in vitro transamidase assay. The formation of this disulfide bond is not essential but required for full transamidase activity. Antibodies against GPI8 and PIG-T revealed that endogenous as well as exogenous proteins formed a disulfide bond. Furthermore trypanosome GPI8 forms a similar intermolecular disulfide bond via its conserved cysteine residue, suggesting that the trypanosome GPI transamidase is also a multimeric complex likely containing the orthologue of PIG-T. We also demonstrate that an inactive human GPI transamidase complex that consists of non-functional GPI8 and four other components was co-purified with the proform of substrate proteins, indicating that these five components are sufficient to hold the substrate proteins.     

Paramecium GPI proteins: variability of expression and localization

In Paramecium primaurelia, the two major classes of cell surface proteins, the surface antigen (SAg) and the surface GPI proteins (SGPs), are linked to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. In the present study, we have characterized the expression of the SGPs in several geographical strains of P. primaurelia and P. tetraurelia at different temperatures, 23 degrees C and 32 degrees C. The identification of the expressed SGPs was performed on purified cilia, by establishing the SGP SDS-PAGE profiles under four different conditions: with or without their anchoring lipid, cleaved with a Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), and either in a reduced or in an unreduced state. This screening revealed the existence of specific sets of ciliary SGPs, as a function of temperature and the geographical origin of the strains. The SGPs the most abundant at 23 degrees C and 32 degrees C displayed a rapid turnover. We also looked for the presence of PI-PLC releasable proteins in purified cortices. In addition to the SAg and SGPs, the cortical fraction was shown to contain other PI-PLC releasable proteins, not found in the ciliary fraction, thus localized exclusively in the interciliary region.     

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.     

The human GPI1 gene is required for efficient glycosylphosphatidylinositol biosynthesis.

The first step in glycosylphosphatidylinositol (GPI) membrane anchor biosynthesis that is defective in paroxysmal nocturnal haemoglobinuria is mediated by an N-acetylglucosaminyl transferase expressed in the endoplasmic reticulum. Six human genes encode subunits of this enzyme, namely PIG-A, PIG-C, PIG-H, PIG-P, GPI1, and DPM2. Here, the human GPI1 gene is characterised. This gene is organised into eleven exons. The locus was mapped to chromosome 16p13.3 near the haemoglobin alpha chain locus. GPI1 is expressed ubiquitously in human cells and tissues. Expression levels are markedly elevated in haematopoietic tissues (bone marrow, foetal liver). To determine whether human GPI1 is essential for human GPI biosynthesis, antisense RNA was expressed in HEK293 cells. Transfectants exhibited a marked but incomplete decrease in the expression of a GPI-linked reporter protein, confirming that GPI1 is required for efficient GPI biosynthesis. In contrast, expression of GPI-linked proteins is normal in lymphatic cell lines from individuals with the alpha thalassaemia/mental retardation syndrome, which is characterised by large deletions from chromosome 16p removing one of the two GPI1 alleles along with the haemoglobin alpha locus. In conclusion, GPI1 plays an important role in the biosynthesis of GPI intermediates. Due to its autosomal localisation, the heterozygous deletion of GPI1 does not lead to an overt defect in the expression of GPI-linked proteins.     

Synthesis of the essential core of the human glycosylphosphatidylinositol (GPI) anchor

The biological role of GPI anchors is of paramount importance; however, we are still far from fully understanding the structure-function relationship of these molecules. One major limiting factor has been the tiny quantities available from natural sources; obtaining homogeneous and well-defined GPI structures by synthesis, is both a challenge and an attractive goal. We report here the convergent synthesis of the essential core of the human GPI anchor 1, exploiting a common precursor to obtain the trisaccharidic donor 2 and a novel protecting groups sequence. The final product, prepared for the first time, is biologically active.     

Gpi19, the Saccharomyces cerevisiae homologue of mammalian PIG-P, is a subunit of the initial enzyme for glycosylphosphatidylinositol anchor biosynthesis

Glycosylphosphatidylinositols (GPIs) are attached to the C termini of some glycosylated secretory proteins, serving as membrane anchors for many of those on the cell surface. Biosynthesis of GPIs is initiated by the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol. This reaction is carried out at the endoplasmic reticulum (ER) by an enzyme complex called GPI-N-acetylglucosaminyltransferase (GPI-GlcNAc transferase). The human enzyme has six known subunits, at least four of which, GPI1, PIG-A, PIG-C, and PIG-H, have functional homologs in the budding yeast Saccharomyces cerevisiae. The uncharacterized yeast gene YDR437w encodes a protein with some sequence similarity to human PIG-P, a fifth subunit of the GPI-GlcNAc transferase. Here we show that Ydr437w is a small but essential subunit of the yeast GPI-GlcNAc transferase, and we designate its gene GPI19. Similar to other mutants in the yeast enzyme, temperature-sensitive gpi19 mutants display cell wall defects and hyperactive Ras phenotypes. The Gpi19 protein associates with the yeast GPI-GlcNAc transferase in vivo, as judged by coimmuneprecipitation with the Gpi2 subunit. Moreover, conditional gpi19 mutants are defective for GPI-GlcNAc transferase activity in vitro. Finally, we present evidence for the topology of Gpi19 within the ER membrane.