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Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii, is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in Toxoplasma gondii has remained largely unknown without understanding its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. By contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which binds GIPLs, exhibits an enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that Δpigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by Δpigj early in the infection in wild-type mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with Δpigj parasites indicate an advantage over wild-type strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcomes in the infected host.IMPORTANCEThe functional significance of sidechain modifications to the glycosylphosphatidylinositol (GPI) anchor in parasites has yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both Toxoplasmsa gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased host susceptibility to infection. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on disease outcomes. This indicates GPI sidechains are fundamental to host-parasite interactions.

Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R2 = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases.IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.

Reversing brain aging may be possible through systemic interventions such as exercise. We found that administration of circulating blood factors in plasma from exercised aged mice transferred the effects of exercise on adult neurogenesis and cognition to sedentary aged mice. Plasma concentrations of glycosylphosphatidylinositol (GPI)-specific phospholipase D1 (Gpld1), a GPI-degrading enzyme derived from liver, were found to increase after exercise and to correlate with improved cognitive function in aged mice, and concentrations of Gpld1 in blood were increased in active, healthy elderly humans. Increasing systemic concentrations of Gpld1 in aged mice ameliorated age-related regenerative and cognitive impairments by altering signaling cascades downstream of GPI-anchored substrate cleavage. We thus identify a liver-to-brain axis by which blood factors can transfer the benefits of exercise in old age.

Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections.

The Babesia divergens/B. capreoli group includes parasites with confirmed or possible zoonotic potential to cause human babesiosis. Currently, diagnostic antigen of the group has not been established. In this study, we investigated the ortholog of Bd37, a glycosylphosphatidylinositol (GPI)-anchored major merozoite surface protein of B. divergens sensu stricto, in the Asia lineage of the group. From two genomic isolates from sporozoite/sporoblast stages, three Bd37 gene variants, namely Bd37 JP-A, JP-B, and JP-C, were isolated with 62.3-64.1% amino acid sequence identity. Discriminative blood direct PCR revealed that Bd37 JP-A was encoded in all parasites infecting wild sika deer examined (n=22). While Bd37 JP-B and JP-C genes were randomly detected in 12 and 11 specimens, respectively. Sequencing of all JP-A variants revealed that the gene was polymorphic, with a low ratio of non-synonymous to synonymous substitutions (dN/dS) and that a highly polymorphic region was not related to predicted B-cell epitopes. A recombinant JP-A-based ELISA showed an overall positive rate of 13.9% in sika deer in Japan from north (Hokkaido) to south (Kyushu island) across 24 prefectures (n=360). This positive rate was twice as high as that examined by 18S rRNA-based PCR (6.6%). The geographical trends in infection rates were consistent. This study demonstrated that direct examination was informative for revealing genetic background and selecting antigen candidates. Bd37 orthologs may serve diagnostic purposes in combination with indirect fluorescence assay, which requires biological isolates.

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.

Abstract Background Defects in the glycosylphosphatidylinositol (GPI) biosynthesis pathway can result in a group of congenital disorders of glycosylation known as the inherited GPI deficiencies (IGDs). To date, defects in 22 of the 29 genes in the GPI biosynthesis pathway have been identified in IGDs. The early phase of the biosynthetic pathway assembles the GPI anchor (Synthesis stage) and the late phase transfers the GPI anchor to a nascent peptide in the endoplasmic reticulum (ER) (Transamidase stage), stabilizes the anchor in the ER membrane using fatty acid remodeling and then traffics the GPI-anchored protein to the cell surface (Remodeling stage). Results We addressed the hypothesis that disease-associated variants in either the Synthesis stage or Transamidase+Remodeling-stage GPI pathway genes have distinct phenotypic spectra. We reviewed clinical data from 58 publications describing 152 individual patients and encoded the phenotypic information using the Human Phenotype Ontology (HPO). We showed statistically significant differences between the Synthesis and Transamidase+Remodeling Groups in the frequencies of phenotypes in the musculoskeletal system, cleft palate, nose phenotypes, and cognitive disability. Finally, we hypothesized that phenotypic defects in the IGDs are likely to be at least partially related to defective GPI anchoring of their target proteins. Twenty-two of one hundred forty-two proteins that receive a GPI anchor are associated with one or more Mendelian diseases and 12 show some phenotypic overlap with the IGDs, represented by 34 HPO terms. Interestingly, GPC3 and GPC6, members of the glypican family of heparan sulfate proteoglycans bound to the plasma membrane through a covalent GPI linkage, are associated with 25 of these phenotypic abnormalities. Conclusions IGDs associated with Synthesis and Transamidase+Remodeling stages of the GPI biosynthesis pathway have significantly different phenotypic spectra. GPC2 and GPC6 genes may represent a GPI target of general disruption to the GPI biosynthesis pathway that contributes to the phenotypes of some IGDs.

PIGT-CDG, an autosomal recessive syndromic intellectual disability disorder of glycosylphosphatidylinositol (GPI) anchors, was recently described in two independent kindreds [Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 3 (OMIM, #615398)]. PIGT encodes phosphatidylinositol-glycan biosynthesis class T, a subunit of the heteropentameric transamidase complex that facilitates the transfer of GPI to proteins. GPI facilitates attachment (anchoring) of proteins to cell membranes. We describe, at ages 7 and 6 years, two children of non-consanguineous parents; they had hypotonia, severe global developmental delay, and intractable seizures along with endocrine, ophthalmologic, skeletal, hearing, and cardiac anomalies. Exome sequencing revealed that both siblings had compound heterozygous variants in PIGT (NM_015937.5), i.e., c.918dupC, a novel duplication leading to a frameshift, and c.1342C > T encoding a previously described missense variant. Flow cytometry studies showed decreased surface expression of GPI-anchored proteins on granulocytes, consistent with findings in previous cases. These siblings further delineate the clinical spectrum of PIGT-CDG, reemphasize the neuro-ophthalmologic presentation, clarify the endocrine features, and add hypermobility, low CSF albumin quotient, and hearing loss to the phenotypic spectrum. Our results emphasize that GPI anchor-related congenital disorders of glycosylation (CDGs) should be considered in subjects with early onset severe seizure disorders and dysmorphic facial features, even in the presence of a normal carbohydrate-deficient transferrin pattern and N-glycan profiling. Currently available screening for CDGs will not reliably detect this family of disorders, and our case reaffirms that the use of flow cytometry and genetic testing is essential for diagnosis in this group of disorders.

Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface.

Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface.

Mutations in the phosphatidylinositol glycan biosynthesis class A (PIGA) gene cause a rare, X-linked recessive congenital disorder of glycosylation. Phosphatidylinositol glycan biosynthesis class A congenital disorder of glycosylation (PIGA-CDG) is characterized by seizures, intellectual and developmental delay, and congenital malformations. The PIGA gene encodes an enzyme involved in the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis. There are over 100 GPI-anchored proteins that attach to the cell surface and are involved in cell signaling, immunity, and adhesion. Little is known about the pathophysiology of PIGA-CDG. Here, we describe the first Drosophila model of PIGA-CDG and demonstrate that loss of PIG-A function in Drosophila accurately models the human disease. As expected, complete loss of PIG-A function is larval lethal. Heterozygous null animals appear healthy but, when challenged, have a seizure phenotype similar to what is observed in patients. To identify the cell-type specific contributions to disease, we generated neuron- and glia-specific knockdown of PIG-A. Neuron-specific knockdown resulted in reduced lifespan and a number of neurological phenotypes but no seizure phenotype. Glia-knockdown also reduced lifespan and, notably, resulted in a very strong seizure phenotype. RNA sequencing analyses demonstrated that there are fundamentally different molecular processes that are disrupted when PIG-A function is eliminated in different cell types. In particular, loss of PIG-A in neurons resulted in upregulation of glycolysis, but loss of PIG-A in glia resulted in upregulation of protein translation machinery. Here, we demonstrate that Drosophila is a good model of PIGA-CDG and provide new data resources for future study of PIGA-CDG and other GPI anchor disorders., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Background: Defects in the glycosylphosphatidylinositol (GPI) biosynthesis pathway can result in a group of congenital disorders of glycosylation known as the inherited GPI deficiencies (IGDs). To date, defects in 22 of the 29 genes in the GPI biosynthesis pathway have been identified in IGDs. The early phase of the biosynthetic pathway assembles the GPI anchor (Synthesis stage) and the late phase transfers the GPI anchor to a nascent peptide in the endoplasmic reticulum (ER) (Transamidase stage), stabilizes the anchor in the ER membrane using fatty acid remodeling and then traffics the GPI-anchored protein to the cell surface (Remodeling stage).Results: We addressed the hypothesis that disease-associated variants in either the Synthesis stage or Transamidase+Remodeling-stage GPI pathway genes have distinct phenotypic spectra. We reviewed clinical data from 58 publications describing 152 individual patients and encoded the phenotypic information using the Human Phenotype Ontology (HPO). We showed statistically significant differences between the Synthesis and Transamidase+Remodeling Groups in the frequencies of phenotypes in the musculoskeletal system, cleft palate, nose phenotypes, and cognitive disability. Finally, we hypothesized that phenotypic defects in the IGDs are likely to be at least partially related to defective GPI anchoring of their target proteins. Twenty-two of one hundred forty-two proteins that receive a GPI anchor are associated with one or more Mendelian diseases and 12 show some phenotypic overlap with the IGDs, represented by 34 HPO terms. Interestingly, GPC3 and GPC6, members of the glypican family of heparan sulfate proteoglycans bound to the plasma membrane through a covalent GPI linkage, are associated with 25 of these phenotypic abnormalities.Conclusions: IGDs associated with Synthesis and Transamidase+Remodeling stages of the GPI biosynthesis pathway have significantly different phenotypic spectra. GPC2 and GPC6 genes may represent a GPI target of general disruption to the GPI biosynthesis pathway that contributes to the phenotypes of some IGDs. [ABSTRACT FROM AUTHOR]

The oligomerization of glycosylphosphatidylinositol-anchored proteins is thought to regulate their association with membrane microdomains, subcellular sorting, and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in human embryo kidnay 293 (HEK293) cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, the diffusion coefficient decreased in monomer-enriched fractions only for the active receptor, suggesting that uPAR monomers might be preferentially engaged in multiprotein transmembrane signaling complexes. Our approach helps in limiting the alteration of the data due to out-of-focus effects and in minimizing the overestimation of the molecular brightness. In addition to a careful design of the cellular model, it gives reliable estimates of diffusion coefficients and oligomerization of GPI-anchored proteins, in steady-state conditions, at low expression levels, and in live, unperturbed cells.

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer-oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein-uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA-plasminogen activator inhibitor type 1-mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents.

T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR-CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.

Endoplasmic reticulum-associated degradation (ERAD) plays indispensable roles in many physiological processes; however, the nature of endogenous substrates remains largely elusive. Here we report a proteomics strategy based on the intrinsic property of the SEL1L-HRD1 ERAD complex to identify endogenous ERAD substrates both in vitro and in vivo. Following stringent filtering using a machine learning algorithm, over 100 high-confidence potential substrates are identified in human HEK293T and mouse brown adipose tissue, among which ~88% are cell type-specific. One of the top shared hits is the catalytic subunit of the glycosylphosphatidylinositol (GPI)-transamidase complex, PIGK. Indeed, SEL1L-HRD1 ERAD attenuates the biogenesis of GPI-anchored proteins by specifically targeting PIGK for proteasomal degradation. Lastly, several PIGK disease variants in inherited GPI deficiency disorders are also SEL1L-HRD1 ERAD substrates. This study provides a platform and resources for future effort to identify proteome-wide endogenous substrates in vivo, and implicates SEL1L-HRD1 ERAD in many cellular processes including the biogenesis of GPI-anchored proteins., (© 2024. The Author(s).)

Folate receptor α (FR) was discovered many decades ago, along with drugs that target intracellular folate metabolism, such as pemetrexed and methotrexate. Folate is taken up by the cell via this receptor, which also targeted by many cancer agents due to the over-expression of the receptor by cancer cells. FR is a membrane-bound glycosyl-phosphatidylinositol (GPI) anchor glycoprotein encoded by the folate receptor 1 (FOLR1) gene. FR plays a significant role in DNA synthesis, cell proliferation, DNA repair, and intracellular signaling, all of which are essential for tumorigenesis. FR is more prevalent in cancer cells compared to normal tissues, which makes it an excellent target for oncologic therapeutics. FRα is found in many cancer types, including ovarian cancer, non-small-cell lung cancer (NSCLC), and colon cancer. FR is widely used in antibody drug conjugates, small-molecule-drug conjugates, and chimeric antigen-receptor T cells. Current oncolytic therapeutics include mirvetuximab soravtansine, and ongoing clinical trials are underway to investigate chimeric antigen receptor T cells (CAR-T cells) and vaccines. Additionally, FRα has been used in a myriad of other applications, including as a tool in the identification of tumor types, and as a prognostic marker, as a surrogate of chemotherapy resistance. As such, FRα identification has become an essential part of precision medicine.

Climate change is a change in the usual weather found in a place. The climate change has a major impact not only on natural disasters of the Earth but also on human health. The climate crisis is then no longer a future concern. It includes both the global warming driven by human emissions of greenhouse gases (GHG), and the resulting large-scale shifts in weather patterns. Global warming can occur from a variety of causes, both natural and human induced. The primary GHG in Earth's atmosphere, listed in decreasing order of average global mole fraction, are: water vapor (H 2 O), carbon dioxide (CO 2 ), methane (CH 4 ), nitrous oxide (N 2 O), and ozone (O 3 ). Today, scientists around the world continue to try and solve the puzzle of climate change. It is clear that to address climate change, the amount of CO 2 released into the atmosphere by industrial process has to be reduced because once it is added to the atmosphere, it can continue to affect climate for thousands of years. For such a purpose, an approach to intervention using expression vectors for any protein targeting to the cell plasma membrane via the glycosylphosphatidylinositol, GPI, anchor is suggested. The resulting GPI-anchored proteins would be useful for studying intermolecular interactions, especially gene-environment interactions, in investigating the potential impact of any chemical compounds on any genes of interest and could be used for carbonic anhydrase (CA)-based CO 2 -capture (environmental application). This approach would be crucial not only for capturing CO 2 via GPI and CA but also for the production of CA enzyme as well as its stabilization and therefore useful for combating the global warming of climate change.

The unfolded protein response (UPR) is an intracellular signaling pathway that relays signals from the lumen of the ER to activate target genes in the nucleus. We devised a genetic screen in the yeast Saccharomyces cerevisiae to isolate mutants that are dependent on activation of the pathway for viability. Using this strategy, we isolated mutants affecting various aspects of ER function, including protein translocation, folding, glycosylation, glycosylphosphatidylinositol modification, and ER-associated protein degradation (ERAD). Extending results gleaned from the genetic studies, we demonstrate that the UPR regulates trafficking of proteins at the translocon to balance the needs of biosynthesis and ERAD. The approach also revealed connections of the UPR to other regulatory pathways. In particular, we identified SON1/RPN4, a recently described transcriptional regulator for genes encoding subunits of the proteasome. Our genetic strategy, therefore, offers a powerful means to provide insight into the physiology of the UPR and to identify novel genes with roles in many aspects of secretory and membrane protein biogenesis.

Heparan sulfate proteoglycans (HSPGs) play diverse roles in cell recognition, growth, and adhesion. In vitro studies suggest that cell-surface HSPGs act as coreceptors for heparin-binding mitogenic growth factors. Here we show that the glycosylphosphatidylinositol- (GPI-) anchored HSPG glypican-1 is strongly expressed in human pancreatic cancer, both by the cancer cells and the adjacent fibroblasts, whereas expression of glypican-1 is low in the normal pancreas and in chronic pancreatitis. Treatment of two pancreatic cancer cell lines, which express glypican-1, with the enzyme phosphoinositide-specific phospholipase-C (PI-PLC) abrogated their mitogenic responses to two heparin-binding growth factors that are commonly overexpressed in pancreatic cancer: fibroblast growth factor 2 (FGF2) and heparin-binding EGF-like growth factor (HB-EGF). PI-PLC did not alter the response to the non-heparin-binding growth factors EGF and IGF-1. Stable expression of a form of glypican-1 engineered to possess a transmembrane domain instead of a GPI anchor conferred resistance to the inhibitory effects of PI-PLC on growth factor responsiveness. Furthermore, transfection of a glypican-1 antisense construct attenuated glypican-1 protein levels and the mitogenic response to FGF2 and HB-EGF. We propose that glypican-1 plays an essential role in the responses of pancreatic cancer cells to certain mitogenic stimuli, that it is relatively unique in relation to other HSPGs, and that its expression by pancreatic cancer cells may be of importance in the pathobiology of this disorder.

After the cellular prion protein (PrPC) transits to the cell surface where it is bound by a glycophosphatidyl inositol (GPI) anchor, PrPC is either metabolized or converted into the scrapie isoform (PrPSc). Because most GPI-anchored proteins are associated with cholesterol-rich membranous microdomains, we asked whether such structures participate in the metabolism of PrPC or the formation of PrPSc. The initial degradation of PrPC involves removal of the NH2 terminus of PrPC to produce a 17-kD polypeptide which was found in a Triton X-100 insoluble fraction. Both the formation of PrPSc and the initial degradation of PrPC were diminished by lovastatin-mediated depletion of cellular cholesterol but were insensitive to NH4Cl. Further degradation of the 17-kD polypeptide did occur within an NH4Cl-sensitive, acidic compartment. Replacing the GPI addition signal with the transmembrane and cytoplasmic domains of mouse CD4 rendered chimeric CD4PrPC soluble in cold Triton X-100. Both CD4PrPC and truncated PrPC without the GPI addition signal (Rogers, M., F. Yehieley, M. Scott, and S. B. Prusiner. 1993. Proc. Natl. Acad. Sci. USA. 90:3182-3186) were poor substrates for PrPSc formation. Thus, it seems likely that both the initial degradation of PrPC to the 17-kD polypeptide and the formation of PrPSc occur within a non-acidic compartment bound by cholesterol-rich membranes, possibly glycolipid-rich microdomains, where the metabolic fate of PrPC is determined. The pathway remains to be identified by which the 17-kD polypeptide and PrPSc are transported to an acidic compartment, presumably endosomes, where the 17-kD polypeptide is hydrolyzed and limited proteolysis of PrPSc produces PrP 27-30.

Cell-surface proteoglycans have been implicated in cell responses to growth factors, extracellular matrix, and cell adhesion molecules. M12, one of the most abundant membrane-associated proteoglycans in the adult rat brain, is a approximately 65 kDa glycosylphosphatidylinositol-linked protein that bears heparan sulfate chains (Herndon and Lander, 1990). To assess its identity, M12 was purified and internal peptide sequences obtained. Comparison of the results with protein sequence predicted by a cDNA cloned from PC12 cells indicated that M12 is rat glypican, a proteoglycan first cloned from human fibroblasts. In addition, antibodies raised against a rat glypican fusion protein specifically detected the 65 kDa brain proteoglycan core protein, both by immunoprecipitation and by Western blotting. Northern blot analysis using a rat glypican probe also detected glypican message in the adult, as well as the developing rat brain. In situ hybridization with glypican RNA probes showed that glypican is expressed in a subset of structures in the adult rat nervous system. These include the hippocampus, dorsal thalamus, amygdala, cerebral cortex, piriform cortex, olfactory tubercle, several cranial nerve nuclei, the ventral horn of the spinal cord, and the dorsal root ganglia. Several other brain regions exhibited little or no hybridization over background. In most cases where glypican hybridization was observed, the signal could be localized specifically to the cell bodies of identifiable neurons, for example, spinal motoneurons, hippocampal pyramidal cells. In the cerebral cortex, glypican hybridization was found in layers 2/3, 5, and 6, but was missing from 1 and 4. The data suggest that glypican is expressed primarily by subpopulations of projection neurons in the adult rat nervous system.

Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. We report here the cloning and pattern of expression of cerebroglycan, a glycosylphosphatidylinositol (GPI)-anchored HSPG that is found in the developing rat brain (previously referred to as HSPG M13; Herndon, M. E., and A. D. Lander. 1990. Neuron. 4:949-961). The cerebroglycan core protein has a predicted molecular mass of 58.6 kD and five potential heparan sulfate attachment sites. Together with glypican (David, G., V. Lories, B. Decock, P. Marynen, J.-J. Cassiman, and H. Van den Berghe. 1990. J. Cell Biol. 111:3165-3176), it defines a family of integral membrane HSPGs characterized by GPI linkage and conserved structural motifs, including a pattern of 14 cysteine residues that is absolutely conserved. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is expressed in only one tissue: the nervous system. In situ hybridization experiments at several developmental stages strongly suggest that cerebroglycan message is widely and transiently expressed by immature neurons, appearing around the time of final mitosis and disappearing after cell migration and axon outgrowth have been completed. These results suggest that cerebroglycan may fulfill a function related to the motile behaviors of developing neurons.

We use a sensitive biotin polarity assay to survey the surface distribution of glycosyl-phosphatidylinositol (GPI) anchored proteins in five model epithelial cell lines derived from different species (dog, pig, man) and tissues, i.e., kidney (MDCK I, MDCK II, LLC-PK1) and intestine (Caco-2 and SK-CO15). After biotinylation of apical or basolateral surfaces of confluent monolayers grown on polycarbonate filters, GPI-anchored proteins are identified by their shift from a Triton X-114 detergent-rich phase to a detergent-poor phase in the presence of phosphatidylinositol-specific phospholipase C. All GPI-anchored proteins detected (3-9 per cell type, at least 13 different proteins) are found to be apically polarized; no GPI-anchored protein is observed preferentially localized to the basal surface. One of the GPI-anchored proteins is identified as carcinoembryonic antigen (CEA). Survey of MDCK II-RCAr, a mutant cell line with a pleiotropic defect in galactosylation of glycoproteins and glycolipids (that presumably affects GPI anchors) also reveals an apical polarization of all GPI-anchored proteins. In contrast, analysis of MDCK II-ConAr (a mutant cell line with an unknown defect in glycosylation) revealed five GPI-anchored proteins, two of which appeared relatively unpolarized. Our results indicate that the polarized apical distribution of GPI-anchored proteins is highly conserved across species and tissue-type and may depend on glycosylation.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

In mammalian cells, misfolded glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are cleared out of the ER to the Golgi via a constitutive and a stress-inducible pathway called RESET. From the Golgi, misfolded GPI-APs transiently access the cell surface prior to rapid internalization for lysosomal degradation. What regulates the release of misfolded GPI-APs for RESET during steady-state conditions and how this release is accelerated during ER stress is unknown. Using mutants of prion protein or CD59 as model misfolded GPI-APs, we demonstrate that inducing calnexin degradation or upregulating calnexin-binding glycoprotein expression triggers the release of misfolded GPI-APs for RESET. Conversely, blocking protein synthesis dramatically inhibits the dissociation of misfolded GPI-APs from calnexin and subsequent turnover. We demonstrate an inverse correlation between newly synthesized calnexin substrates and RESET substrates that coimmunoprecipitate with calnexin. These findings implicate competition by newly synthesized substrates for association with calnexin as a key factor in regulating the release of misfolded GPI-APs from calnexin for turnover via the RESET pathway., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Many eukaryotic receptors and enzymes rely on glycosylphosphatidylinositol (GPI) anchors for membrane localization and function. The transmembrane complex GPI-T recognizes diverse proproteins at a signal peptide region that lacks consensus sequence and replaces it with GPI via a transamidation reaction. How GPI-T maintains broad specificity while preventing unintentional cleavage is unclear. Here, substrates- and products-bound human GPI-T structures identify subsite features that enable broad proprotein specificity, inform catalytic mechanism, and reveal a multilevel safeguard mechanism against its promiscuity. In the absence of proproteins, the catalytic site is invaded by a locally stabilized loop. Activation requires energetically unfavorable rearrangements that transform the autoinhibitory loop into crucial catalytic cleft elements. Enzyme-proprotein binding in the transmembrane and luminal domains respectively powers the conformational rearrangement and induces a competent cleft. GPI-T thus integrates various weak specificity regions to form strong selectivity and prevent accidental activation. These findings provide important mechanistic insights into GPI-anchored protein biogenesis., (© 2023. Springer Nature Limited.)

ABSTRACT Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that infects humans and animals. The pathogenesis of T. gondii is linked to its lytic cycle, which starts when tachyzoites invade host cells and secrete proteins from specialized organelles. Once inside the host cell, the parasite creates a parasitophorous vacuole (PV) where it divides. Rhoptries are specialized secretory organelles that contain proteins, many of which are secreted during invasion. These proteins have important roles not only during the initial interaction between parasite and host but also in the formation of the PV and in the modification of the host cell. We report here the identification of a new T. gondii carbonic anhydrase-related protein (TgCA_RP), which localizes to rhoptries of mature tachyzoites. TgCA_RP is important for the morphology of rhoptries and for invasion and growth of parasites. TgCA_RP is also critical for parasite virulence. We propose that TgCA_RP plays a role in the biogenesis of rhoptries.