Glycosylphosphatidylinositol Transamidase Complex Research Articles

Spectrum of Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 3 (MCAHS3) Due to Phosphatidylinositol Glycan Biosynthesis Class T (PIGT) Gene Mutations: A Narrative Review.

Multiple congenital anomalies-hypotonia-seizures syndrome 3 (MCAHS3) results from mutations in the phosphatidylinositol glycan biosynthesis class T (PIGT) gene leading to defects in glycosylphosphatidylinositol transamidase complex (GPI-TA) synthesis. Glycosylphosphatidylinositol serves as an anchor to more than 150 mammalian proteins for attachment on cell surfaces, enabling specific functional properties. Mutations in the PIGT gene result in disruption of this extremely important post-translational protein modification, yielding dysfunctional proteins leading to MCAHS3. An exhaustive literature search was conducted across various electronic databases to reveal only 41 reported cases of MCAHS3 worldwide, emphasizing the rarity of this condition. Multiple congenital anomalies-hypotonia-seizures syndrome 3 has been reported as secondary to 18 different known PIGT variants to date, manifesting as a varying spectrum of craniofacial dysmorphism, developmental delay with epilepsy, cardiac and renal malformations, and unique features in biochemical testing and neuroimaging. This review aims to highlight the constellation of clinical symptoms, diagnostic modalities, and management challenges associated with MCAHS3 cases. It would help determine optimal diagnostic and treatment strategies for newly identified cases and facilitate new research on this rare condition.

Structure and Function of the Glycosylphosphatidylinositol Transamidase, a Transmembrane Complex Catalyzing GPI Anchoring of Proteins.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a ubiquitous posttranslational modification in eukaryotic cells. GPI-anchored proteins (GPI-APs) play critical roles in enzymatic, signaling, regulatory, and adhesion processes. Over 20 enzymes are involved in GPI synthesis, attachment to client proteins, and remodeling after attachment. The GPI transamidase (GPI-T), a large complex located in the endoplasmic reticulum membrane, catalyzes the attachment step by replacing a C-terminal signal peptide of proproteins with GPI. In the last three decades, extensive research has been conducted on the mechanism of the transamidation reaction, the components of the GPI-T complex, the role of each subunit, and the substrate specificity. Two recent studies have reported the three-dimensional architecture of GPI-T, which represent the first structures of the pathway. The structures provide detailed mechanisms for assembly that rationalizes previous biochemical results and subunit-dependent stability data. While the structural data confirm the catalytic role of PIGK, which likely uses a caspase-like mechanism to cleave the proproteins, they suggest that unlike previously proposed, GPAA1 is not a catalytic subunit. The structures also reveal a shared cavity for GPI binding. Somewhat unexpectedly, PIGT, a single-pass membrane protein, plays a crucial role in GPI recognition. Consistent with the assembly mechanisms and the active site architecture, most of the disease mutations occur near the active site or the subunit interfaces. Finally, the catalytic dyad is located ~22Å away from the membrane interface of the GPI-binding site, and this architecture may confer substrate specificity through topological matching between the substrates and the elongated active site. The research conducted thus far sheds light on the intricate processes involved in GPI anchoring and paves the way for further mechanistic studies of GPI-T.

CLPTM1L is a GPI-anchoring pathway component targeted by HCMV.

The GPI-anchoring pathway plays important roles in normal development and immune modulation. MHC Class I Polypeptide-related Sequence A (MICA) is a stress-induced ligand, downregulated by human cytomegalovirus (HCMV) to escape immune recognition. Its most prevalent allele, MICA*008, is GPI-anchored via an uncharacterized pathway. Here, we identify cleft lip and palate transmembrane protein 1-like protein (CLPTM1L) as a GPI-anchoring pathway component and show that during infection, the HCMV protein US9 downregulates MICA*008 via CLPTM1L. We show that the expression of some GPI-anchored proteins (CD109, CD59, and MELTF)-but not others (ULBP2, ULBP3)-is CLPTM1L-dependent, and further show that like MICA*008, MELTF is downregulated by US9 via CLPTM1L during infection. Mechanistically, we suggest that CLPTM1L's function depends on its interaction with a free form of PIG-T, normally a part of the GPI transamidase complex. We suggest that US9 inhibits this interaction and thereby downregulates the expression of CLPTM1L-dependent proteins. Altogether, we report on a new GPI-anchoring pathway component that is targeted by HCMV.

Molecular insights into biogenesis of glycosylphosphatidylinositol anchor proteins

Eukaryotic cells are coated with an abundance of glycosylphosphatidylinositol anchor proteins (GPI-APs) that play crucial roles in fertilization, neurogenesis, and immunity. The removal of a hydrophobic signal peptide and covalent attachment of GPI at the new carboxyl terminus are catalyzed by an endoplasmic reticulum membrane GPI transamidase complex (GPI-T) conserved among all eukaryotes. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GPI-T at a global 2.53-Å resolution, revealing an equimolar heteropentameric assembly. Structure-based mutagenesis suggests a legumain-like mechanism for the recognition and cleavage of proprotein substrates, and an endogenous GPI in the structure defines a composite cavity for the lipid substrate. This elongated active site, stemming from the membrane and spanning an additional ~22-Å space toward the catalytic dyad, is structurally suited for both substrates which feature an amphipathic pattern that matches this geometry. Our work presents an important step towards the mechanistic understanding of GPI-AP biosynthesis.

Structure of human glycosylphosphatidylinositol transamidase.

Glycosylphosphatidylinositol (GPI) molecules are complex glycophospholipids and serve as membrane anchors for tethering many proteins to the cell surface. Attaching GPI to the protein in the endoplasmic reticulum (ER) is catalyzed by the transmembrane GPI transamidase (GPIT) complex, which is essential for maturation of the GPI-anchored proteins. The GPIT complex is known to be composed of five subunits: PIGK, PIGU, PIGT, PIGS and GPAA1. Here, we determined the structure of the human GPIT complex at a resolution of 3.1 Å using single-particle cryo-EM, elucidating its overall assembly. The PIGK subunit functions as the catalytic component, in which we identified a C206-H164-N58 triad that is critical for the transamination reaction. Transmembrane helices constitute a widely opened cleft, which is located underneath PIGK, serving as a GPI substrate-binding site. The ubiquitin E3 ligase RNF121 is visualized at the back of the complex and probably serves as a quality control factor for the GPIT complex.

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit.

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Subunits of the GPI transamidase complex localize to the endoplasmic reticulum and nuclear envelope in Drosophila.

A total of 10-20% of plasma membrane proteins are anchored by glycosylphosphatidylinositol (GPI). GPI is attached to proteins by GPI transamidase (GPI-T), which contains five subunits named PIGK, PIGS, PIGT, PIGU, and GPAA1. We previously reported that PIGT localizes near the nucleus in Drosophila. However, localizations of the other four subunits remain unknown. Here, we show that a catalytic subunit of GPI-T, PIGK, mainly localizes to the endoplasmic reticulum (ER), while the other four subunits localize to the nuclear envelope (NE) and ER. The NE/ER localization ratio of PIGS differs between cell types and developmental stages. Our results suggest that GPI-T catalyzes GPI attachment in the ER and the other four subunits may have other unknown functions in the NE.

Hrd1-dependent Degradation of the Unassembled PIGK Subunit of the GPI Transamidase Complex.

Glycosylphosphatidylinositol (GPI)-anchored proteins are post-transcriptionally modified with GPI and anchored to the plasma membrane. GPI is attached to nascent proteins in the endoplasmic reticulum by the GPI transamidase complex, which consists of PIGT, PIGK, GPAA1, PIGU, and PIGS. Of these, PIGK is a catalytic subunit that is unstable without PIGT. This study investigated the pathway by which unassembled PIGK not incorporated into the complex is degraded. We showed that unassembled PIGK was degraded via the proteasome-dependent pathway and that Hrd1 (also known as SYVN1), a ubiquitin ligase involved in the endoplasmic reticulum-associated degradation pathway, was responsible for degradation of unassembled PIGK.Key words: Glycosylphosphatidylinositol, GPI transamidase complex, protein stability, transamidation, ERAD.

AtPIG-S, a predicted Glycosylphosphatidylinositol Transamidase subunit, is critical for pollen tube growth in Arabidopsis

BackgroundGlycosylphosphatidylinositol (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 AtGPI8 (gpi8–2), 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, gpi8–2 almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that gpi8–2 FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized.ResultsHere we investigated the effect of loss of AtPIG-S, another GPI-T subunit, in both gametophytes. Like gpi8–2, we showed that a mutation in AtPIG-S (pigs-1) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG. Still, pigs-1 is transmitted normally through the FG. Conversely, pigs-1 severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A pPIGS:GFP-PIGS transgene complemented these MG defects and enabled generation of pigs-1/pigs-1 seedlings. However, the pPIGS:GFP-PIGS transgene seemingly failed to rescue the function of AtPIG-S in the sporophyte, as pigs-1/pigs-1, pPIGS:GFP-PIGS seedlings died soon after germination.ConclusionsCharacterization of pigs-1 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. Pigs-1 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 pigs-1 is defective only in the downstream transamidation step.

A novel variant in GPAA1, encoding a GPI transamidase complex protein, causes inherited vascular anomalies with various phenotypes.

Vascular anomalies (VAs), comprising wide subtypes of tumors and malformations, are often caused by variants in multiple tyrosine kinase (TK) receptor signaling pathways including TIE2, PIK3CA and GNAQ/11. Yet, a portion of individuals with clinical features of VA do not have variants in these genes, suggesting that there are undiscovered pathogenic factors underlying these patients and possibly with overlapping phenotypes. Here, we identified one rare non-synonymous variant (c.968A > G) in the seventh exon of GPAA1 (Glycosylphosphatidylinositol Anchor Attachment Protein 1), shared by the four affected members of a large pedigree with multiple types of VA using whole-exome sequencing. GPAA1 encodes a glycosylphosphatidylinositol (GPI) transamidase complex protein. This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum (ER). We showed such variant led to scarce expression of GPAA1 protein in vascular endothelium and induced a localization change from ER membrane to cytoplasm and nucleus. In addition, expressing wild-type GPAA1 in endothelial cells had an effect to inhibit cell proliferation and migration, while expressing variant GPAA1 led to overgrowth and overmigration, indicating a loss of the quiescent status. Finally, a gpaa1-deficient zebrafish model displayed several types of developmental defects as well as vascular dysplasia, demonstrating that GPAA1 is involved in angiogenesis and vascular remodeling. Altogether, our results indicate that the rare coding variant in GPAA1 (c.968A > G) is causally related to familial forms of VAs.

Bi-allelic Variants in the GPI Transamidase Subunit PIGK Cause a Neurodevelopmental Syndrome with Hypotonia, Cerebellar Atrophy, and Epilepsy

Glycosylphosphatidylinositol (GPI)-anchored proteins are critical for embryogenesis, neurogenesis, and cell signaling. Variants in several genes participating in GPI biosynthesis and processing lead to decreased cell surface presence of GPI-anchored proteins (GPI-APs) and cause inherited GPI deficiency disorders (IGDs). In this report, we describe 12 individuals from nine unrelated families with 10 different bi-allelic PIGK variants. PIGK encodes a component of the GPI transamidase complex, which attaches the GPI anchor to proteins. Clinical features found in most individuals include global developmental delay and/or intellectual disability, hypotonia, cerebellar ataxia, cerebellar atrophy, and facial dysmorphisms. The majority of the individuals have epilepsy. Two individuals have slightly decreased levels of serum alkaline phosphatase, while eight do not. Flow cytometric analysis of blood and fibroblasts from affected individuals showed decreased cell surface presence of GPI-APs. The overexpression of wild-type (WT) PIGK in fibroblasts rescued the levels of cell surface GPI-APs. In a knockout cell line, transfection with WT PIGK also rescued the GPI-AP levels, but transfection with the two tested mutant variants did not. Our study not only expands the clinical and known genetic spectrum of IGDs, but it also expands the genetic differential diagnosis for cerebellar atrophy. Given the fact that cerebellar atrophy is seen in other IGDs, flow cytometry for GPI-APs should be considered in the work-ups of individuals presenting this feature.

Mutations in PIGU Impair the Function of the GPI Transamidase Complex, Causing Severe Intellectual Disability, Epilepsy, and Brain Anomalies

The glycosylphosphatidylinositol (GPI) anchor links over 150 proteins to the cell surface and is present on every cell type. Many of these proteins play crucial roles in neuronal development and function. Mutations in 18 of the 29 genes implicated in the biosynthesis of the GPI anchor have been identified as the cause of GPI biosynthesis deficiencies (GPIBDs) in humans. GPIBDs are associated with intellectual disability and seizures as their cardinal features. An essential component of the GPI transamidase complex is PIGU, along with PIGK, PIGS, PIGT, and GPAA1, all of which link GPI-anchored proteins (GPI-APs) onto the GPI anchor in the endoplasmic reticulum (ER). Here, we report two homozygous missense mutations (c.209T>A [p.Ile70Lys] and c.1149C>A [p.Asn383Lys]) in five individuals from three unrelated families. All individuals presented with global developmental delay, severe-to-profound intellectual disability, muscular hypotonia, seizures, brain anomalies, scoliosis, and mild facial dysmorphism. Using multicolor flow cytometry, we determined a characteristic profile for GPI transamidase deficiency. On granulocytes this profile consisted of reduced cell-surface expression of fluorescein-labeled proaerolysin (FLAER), CD16, and CD24, but not of CD55 and CD59; additionally, B cells showed an increased expression of free GPI anchors determined by T5 antibody. Moreover, computer-assisted facial analysis of different GPIBDs revealed a characteristic facial gestalt shared among individuals with mutations in PIGU and GPAA1. Our findings improve our understanding of the role of the GPI transamidase complex in the development of nervous and skeletal systems and expand the clinical spectrum of disorders belonging to the group of inherited GPI-anchor deficiencies.

Mutations in GPAA1, Encoding a GPI Transamidase Complex Protein, Cause Developmental Delay, Epilepsy, Cerebellar Atrophy, and Osteopenia

Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs∗102] and c.920delG [p.Gly307Alafs∗11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system.

Post-translational Regulation of Radioactive Iodine Therapy Response in Papillary Thyroid Carcinoma.

Radioactive iodine (RAI) is the mainstay of treatment for differentiated thyroid carcinoma (DTC). Nevertheless, the mechanism of RAI resistance that occurs in many patients with DTC remains unknown. We aimed to elucidate the role of post-translational regulation of radioiodine uptake. We analyzed the expression pattern of the ribosomal glycosylphosphatidylinositol transamidase (GPIT) complex in freshly excised tumors from 10 patients with DTC. We used functional RAI uptake assays to assess the role of GPIT in iodine uptake both in vivo and in vitro. The effects of MEK inhibition on the GPIT subunit PIGU and the sodium iodide symporter (NIS) were assessed in three DTC cell lines and in four human DTC biopsies. We used a multivariable logistic regression model to study the role of PIGU in the response to RAI treatment in advanced DTC. All statistical tests were two-sided. Expression profiling of different GPIT complex subunits revealed statistically significantly lower expression of PIGU in papillary carcinomas than in matched normal thyroid tissue (P < .001). Expression of PIGU in the K1 human papillary carcinoma cell line resulted in a robust increase in NIS glycosylation and trafficking to the cell membrane, accompanied by a robust increase in I125 uptake both in vitro (465 200 ± 56 343 vs 1236 ± 156 counts per million, P < .001) and in vivo (128 945 ± 28 556 vs 7963 ± 192 counts per million, P < .001, n = 5 mice per group). Treatment with the MEK inhibitors U0126 and PD302 rescued PIGU expression. Finally, the PIGU expression levels in tumors of 18 patients with recurrent DTC were associated with a biochemical response to RAI treatment (hazard ratio = 8.06, 95% confidence interval = 3.72 to 12.3, P = .001). We showed that downregulation of PIGU in DTC determines NIS function and RAI avidity. This represents a novel mechanism for RAI resistance.

Disulfide Bond Formation and N-Glycosylation Modulate Protein-Protein Interactions in GPI-Transamidase (GPIT)

Glycosylphosphatidylinositol (GPI) transamidase (GPIT), the enzyme that attaches GPI anchors to proteins as they enter the lumen of the endoplasmic reticulum, is a membrane-bound hetero-pentameric complex consisting of Gpi8, Gpi16, Gaa1, Gpi17 and Gab1. Here, we expressed and purified the luminal domain of Saccharomyces cerevisiae (S. cerevisiae) Gpi8 using different expression systems, and examined its interaction with insect cell expressed luminal domain of S. cerevisiae Gpi16. We found that the N-terminal caspase-like domain of Gpi8 forms a disulfide-linked dimer, which is strengthened by N-glycosylation. The non-core domain of Gpi8 following the caspase-like domain inhibits this dimerization. In contrast to the previously reported disulfide linkage between Gpi8 and Gpi16 in human and trypanosome GPIT, our data show that the luminal domains of S. cerevisiae Gpi8 and S. cerevisiae Gpi16 do not interact directly, nor do they form a disulfide bond in the intact S. cerevisiae GPIT. Our data suggest that subunit interactions within the GPIT complex from different species may vary, a feature that should be taken into account in future structural and functional studies.

The Glycosylphosphatidylinositol Anchor Biosynthesis Genes GPI12, GAA1, and GPI8 Are Essential for Cell-Wall Integrity and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of β-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins. While mutagenesis of auxiliary genes of this pathway suggested roles of GPI-anchored proteins in hyphal growth and virulence, essential genes of this pathway have not been characterized. Taking advantage of RNA interference (RNAi) we analyzed the function of the three essential genes GPI12, GAA1 and GPI8, encoding a cytoplasmic N-acetylglucosaminylphosphatidylinositol deacetylase, a metallo-peptide-synthetase and a cystein protease, the latter two representing catalytic components of the GPI transamidase complex. RNAi strains showed drastic cell-wall defects, resulting in exploding infection cells on the plant surface and severe distortion of in planta-differentiated infection hyphae, including formation of intrahyphal hyphae. Reduction of transcript abundance of the genes analyzed resulted in nonpathogenicity. We show here for the first time that the GPI synthesis genes GPI12, GAA1, and GPI8 are indispensable for vegetative development and pathogenicity of the causal agent of maize anthracnose, Colletotrichum graminicola.

Evolutionary engineering of a wine yeast strain revealed a key role of inositol and mannoprotein metabolism during low-temperature fermentation.

BackgroundWine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes.ResultsWe used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 °C. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins.ConclusionsIn this study we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1755-2) contains supplementary material, which is available to authorized users.

Breaking the boundaries of the GPI transamidase complex (LB147)

Breaking the boundaries of the GPI transamidase complex Dilani G. Gamage, and Tamara L. Hendrickson Department of Chemistry, Wayne State University, Detroit, Michigan 48202 Glycosylphosphatidylinositol transamidase (GPI‐T) is a complicated, membrane‐bound, multi‐subunit enzyme that catalyzes an essential post‐translational modification.1‐3 This enzyme attaches GPI anchors to the C‐termini of various proteins that contain a proper GPI‐T signal sequence. Gpi8, Gaa1, Gpi16, Gpi17 and Gab1 are the five known subunits that may encompass the fungal GPI‐T; Gpi8 is the catalytic subunit but the functions of the other subunits remain largely unknown.5,6 In humans, different GPI‐T subunits are upregulated in different cancers, making GPI‐T a target for cancer research.7,8 However, in spite of the importance of this enzyme, little is known about how it assembles into an active enzyme complex, the stoichiometry of this complex, or the roles of the different components. Here we use soluble domains of the three core subunits (Gpi8, Gpi16 and Gaa1) to investigate the stoichiometry of the enzyme as well as to study the functions of each subunit. Results are shown for the homodimerization of the soluble domain of Gpi8 and the effect of N‐linked glycosylation on Gpi8 homodimerization. Co‐immunoprecipitation of the soluble domains of Gpi8:Gaa1, Gpi8:Gpi16 and Gpi8:Gpi16:Gaa1 discloses a minimal arrangement for these subunits in the absence of their transmembrane domains. Our results will help to elucidate the function and resolve the complexity of GPI‐T. To better understand how changes in expression affect GPI‐T activity, and as a model for this enzyme in cancer, we have also developed an in vivo strategy to monitor and quantify the effect of subunit overexpression on cell surface presentation of GPI‐anchored proteins in Saccharomyces cerevisiae.Grant Funding Source: Wayne State University and the American Cancer Society.

Low-resolution structure of the soluble domain GPAA1 (yGPAA170–247) of the glycosylphosphatidylinositol transamidase subunit GPAA1 from Saccharomyces cerevisiae

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA170–247 and yGPAA170–339 of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70–247 and 70–339 respectively. The secondary structural content of the stable and monodisperse yGPAA170–247 has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA170–247 has an Rg (radius of gyration) of 2.72±0.025 nm and Dmax (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA170–247. The large elliptical shape of yGPAA170–247 is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA170–247 will be discussed together with the recently determined low-resolution structures of yPIG-K24–337 and yPIG-S38–467 from S. cerevisiae in the GPI transamidase complex.

Characterization of GPIT-1 and GPIT-2, two auxiliary components of the Neurospora crassa GPI transamidase complex

The glycosylphosphatidylinositol (GPI) transamidase contains five known subunits and functions in the lumen of the ER to produce GPI-anchored proteins. The transamidase cleaves proteins containing a GPI anchor attachment signal at their C terminus and generates an amide bond between the newly generated carboxyl terminus of the protein and a GPI anchor. We have identified and characterized GPIT-1 and GPIT-2, two of the transamidase subunits from Neurospora crassa. GPIT-1 and GPIT-2 are homologs of the human PIG-T and PIG-U transamidase subunits respectively. We demonstrated that GPIT-2 is required for the addition of GPI anchors onto GPI-anchored proteins. We employed the Neurospora RIP (repeat-induced point mutation) phenomenon to generate 106 “noncritical” amino acid changes in GPIT-1 and 84 “noncritical” amino acid changes in GPIT-2. We used the data to evaluate three-dimensional models for the structures of GPIT-1 and GPIT-2. The mutational data for GPIT-1 is consistent with a multiple-blade propeller structure containing a central channel. The mutational analysis for GPIT-2 supports a structural model based on the karyopherin alpha subunit.