Glycosylphosphatidylinositol Research Articles

The First Report of Polymorphisms and Genetic Characteristics of the Shadow of Prion Protein (SPRN) in Prion Disease-Resistant Animal, Chickens

Prion diseases are irreversible neurodegenerative disorders caused by the aggregated form of prion protein (PrPSc) derived from the normal form of prion protein (PrPC). Previous studies have reported that shadow of prion protein (Sho) interacts with prion protein (PrP) and accelerates the conversion of PrPC to PrPSc. In addition, genetic polymorphisms of the shadow of the prion protein gene (SPRN) are related to the vulnerability of prion diseases in various hosts. However, to date, polymorphisms and genetic features of the SPRN gene have not been investigated in chickens, which are prion disease-resistant animals. We investigated genetic polymorphisms of the SPRN gene in 2 breeds of chickens, i.e., Dekalb White and Ross, using amplicon sequencing. We analyzed genotype, allele and haplotype frequencies and linkage disequilibrium (LD) among the genetic polymorphisms. In addition, we compared the amino acid sequences of Sho among several prion-related species to identify the unique genetic features of chicken Sho using ClustalW. Furthermore, we evaluated the N-terminal signal peptide and glycosylphosphatidylinositol (GPI)-anchor using SignalP and PredGPI, respectively. Finally, we compared the number of SPRN polymorphisms between prion disease-resistant and prion disease-susceptible animals. We identified 7 novel single nucleotide polymorphisms (SNPs), including 1 synonymous SNP in the open reading frame (ORF) of the chicken SPRN gene. We also found significantly different genotypes, allele frequencies and haplotypes between the 2 chicken breeds. In addition, we found that the interaction regions between Sho and PrP and the NXT glycosylation motif were conserved among all species. Notably, sequence similarity was extremely low in the N-terminal and C-terminal regions between mammals and chickens. Furthermore, we found that chicken Sho was the longest N-terminal signal peptide, and the amino acids of the cutting site of chicken are different from those of mammals. Last, unlike other species investigated, omega-site and signal sequences of the GPI-anchor were not found in chickens. To the best of our knowledge, this is the first report of genetic polymorphisms of the SPRN gene in chickens.

CD73-Adenosine A1R Axis Regulates the Activation and Apoptosis of Hepatic Stellate Cells Through the PLC-IP3-Ca2+/DAG-PKC Signaling Pathway.

Alcohol-related liver fibrosis (ALF) is a form of alcohol-related liver disease (ALD) that generally occurs in response to heavy long-term drinking. Ecto-5′-nucleotidase (NT5E), also known as CD73, is a cytomembrane protein linked to the cell membrane via a GPI anchor that regulates the conversion of extracellular ATP to adenosine. Adenosine and its receptors are important regulators of the cellular response. Previous studies showed that CD73 and adenosine A1 receptor (A1R) were important in alcohol-related liver disease, however the exact mechanism is unclear. The aim of this study was to elucidate the role and mechanism of the CD73-A1R axis in both a murine model of alcohol and carbon tetrachloride (CCl4) induced ALF and in an in vitro model of fibrosis induced by acetaldehyde. The degree of liver injury was determined by measuring serum AST and ALT levels, H & E staining, and Masson’s trichrome staining. The expression levels of fibrosis indicators and PLC-IP3-Ca2+/DAG-PKC signaling pathway were detected by quantitative real-time PCR, western blotting, ELISA, and calcium assay. Hepatic stellate cell (HSC) apoptosis was detected using the Annexin V-FITC/PI cell apoptosis detection kit. Knockdown of CD73 significantly attenuated the accumulation of α-SMA and COL1a1 damaged the histological architecture of the mouse liver induced by alcohol and CCl4. In vitro, CD73 inhibition attenuated acetaldehyde-induced fibrosis and downregulated A1R expression in HSC-T6 cells. Inhibition of CD73/A1R downregulated the expression of the PLC-IP3-Ca2+/DAG-PKC signaling pathway. In addition, silencing of CD73/A1R promoted apoptosis in HSC-T6 cells. In conclusion, the CD73-A1R axis can regulate the activation and apoptosis of HSCs through the PLC-IP3-Ca2+/DAG-PKC signaling pathway.

Novel Promising Antifungal Target Proteins for Conquering Invasive Fungal Infections.

Invasive fungal infections (IFIs) pose a serious clinical problem, but the antifungal arsenal is limited and has many disadvantages, such as drug resistance and toxicity. Hence, there is an urgent need to develop antifungal compounds that target novel target proteins of pathogenic fungi for treating IFIs. This review provides a comprehensive summary of the biological functions of novel promising target proteins for treating IFIs in pathogenic fungi and their inhibitors. Inhibitors of inositol phosphoramide (IPC) synthases (such as Aureobasidin A, Khafrefungin, Galbonolide A, and Pleofungin A) have potent antifungal activities by inhibiting sphingolipid synthesis. Disrupting glycosylphosphatidylinositol (GPI) biosynthesis by Jawsamycin (an inhibitor of Spt14), M720 (an inhibitor of Mcd4), and APX001A (an inhibitor of Gwt1) is a promising strategy for treating IFIs. Turbinmicin is a natural-compound inhibitor of Sec14 and has extraordinary antifungal efficacy, broad-antifungal spectrum, low toxicity, and is a promising new compound for treating IFIs. CMLD013075 targets fungal heat shock protein 90 (Hsp90) and has remarkable antifungal efficacy. Olorofim, as an inhibitor of dihydrolactate dehydrogenase, is a breakthrough drug treatment for IFIs. These novel target proteins and their inhibitors may overcome the limitations of currently available antifungal drugs and improve patient outcomes in the treatment of IFIs.

A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag.

Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positive cells, and the TST tag at the N terminal of EGFP allows efficient affinity sorting of the positive cells using Strep-Tactin magnetic beads. The cell sorting system enables efficient isolation of gene transfer-positive cells in a simple, convenient, and fast manner. Cell sorting on transfected K-562 cells resulted in a final positive cell percentage of up to 95.0% with a positive cell enrichment fold of 5.8 times. The applications in gene overexpression experiments could dramatically increase the gene overexpression fold from 10 times to 58 times, and in shRNA gene knockdown experiments, cell sorting increased the gene knockdown efficiency from 12% to 53%. In addition, cell sorting in CRISPR/Cas9 genome editing experiments allowed more significant gene modification, with an editing percentage increasing from 20% to 79%. The gene transfer-positive cell sorting system holds great potential for all gene transfer studies, especially on those hard-to-transfect cells.

Abstract LB540: Potential role of the GPI anchor of Thy-1 in expanding ovarian cancer stem cells

Abstract Introduction: Ovarian cancer is the deadliest gynecologic cancer, and it is often detected at a late stage. Understanding the mechanisms of cancer metastasis can allow us to develop methods to detect ovarian cancers sooner. Screening for specific biomarkers present in at-risk patients may facilitate detection at earlier stages and improve prognoses. A hypothesis currently being developed involves tumor spheroids that infiltrate the abdominal cavity to drive metastasis. These spheroids have increased expression of proteins with a glycosylphosphatidylinositol anchor (GPI anchor). Increased expression of GPI anchored proteins has been previously linked to cancer cells when compared to non-cancerous cells. Thy-1 (CD90) is a cell surface glycoprotein containing a GPI anchor linkage is thought to have a role in forming and increasing proliferation of these cancer spheroids. Methods and Results: In this project, cells were cultured from two ovarian cancer cell lines (A1847 and C30) to determine if the GPI anchor was granting the spheroid proliferation capability as part of the CD90 protein structure. The A1847 and C30 ovarian cancer cell lines were transfected with a lentivirus to express three different versions of the GPI anchor + Thy1 amino acids. Construct I contain truncated Thy-1 protein with the C-terminal 30 amino acids with the GPI anchor, Construct II has truncated Thy-1 (the same C-terminal amino acids as construct I) without the GPI anchor, and Construct III has the full Thy-1 protein and the GPI anchor. The cell lines were grown on non-adherent plates to allow formation of spheroids. Spheroid formation is dependent on the number of cancer stem cells. The size and number of spheroids were measured using microscopy. Spheroids were defined as an aggregate of five or more cells. Spheroid density was calculated using ImageJ from five random fields for each cell line at 2, 24, 48, and 72 hours after seeding onto non-adherent plates. Analysis shows that construct I and construct III have a similar number and density of spheroids indicating that the GPI anchor and the 30 amino acids preceding the anchor are sufficient for promoting spheroid formation. This implies that this may be the region of interest of the Thy-1 protein that can be targeted for diagnosis and treatment. Conclusion: Determining the role of the GPI anchor and the amino acid components of Thy-1 for the self-renewal function of ovarian cancer stem cells is the first step in investigating possible roles for this glycan structure in ovarian cancer metastasis. The results of these studies can lead to future development of diagnostic and therapeutic techniques for earlier detection and treatment of ovarian cancer. Citation Format: Veronica Alvarez, Karen L. Abbott. Potential role of the GPI anchor of Thy-1 in expanding ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB540.

Abstract 93: Soluble mesothelin acts as a signaling molecule

Abstract Mesothelin (MSLN) is a cell surface glycoprotein that is expressed by most pancreatic ductal adenocarcinoma (PDAC) and is used as a target for anti-cancer therapy. MSLN expression appears to make pancreatic cancer more aggressive. Previously, we found that MSLN KO PDAC cells grow in culture and as subcutaneous tumors similarly to wild-type (WT) cells but form intraperitoneal metastases poorly. MSLN pre-cursor (pre-MSLN) is a 71-kD glycosylphosphatidylinositol (GPI)-anchored protein that is cleaved intracellularly to make the 31-kD secreted protein Megakaryocyte Potentiating Factor (MPF), and a 40-kD mature MSLN (mMSLN) that retains membrane attachment. The mMSLN can be shed from the cell surface by the action of specific proteases, resulting in release of soluble MSLN (sMSLN) to both the tumor microenvironment and serum. The goal of this study is to see if sMSLN promotes cancer cell aggressiveness. We transduced MSLN KO pancreas cancer cells to express: 1) a mutant, secreted-only pre-MSLN (includes WT MPF) with a C-terminal truncation (MSLNΔ599) that prevents GPI-linkage of MSLN to the membrane, and 2) truncated pre-MSLN consisting of MPF only. Results show that complementation of KO cells with MPF or MSLNΔ599 does not rescue the MSLN KO phenotype, while MSLNΔ599 bearing a Y318A point mutation that disrupts the binding of MSLN to MUC16/CA125 does restore growth of peritoneal metastasis. We performed RNA-deep sequencing on MSLN KO cells exposed to WT or Y318A sMSLN from conditioned medium. We identified 2000 genes that were differentially expressed across the treatment groups. Exposure to sMSLN resulted in changes in expression of cell adhesion and sterol biosynthetic synthesis pathway genes. Individual genes that were top hits were also identified and are under investigation. Exposure to sMSLN changes the transcriptomic program of pancreas cancer cells and may promote peritoneal colonization. Citation Format: Theressa Ewa, Leela Avula, Sarah Joseph, Michael Rudloff, Samantha Sevilla, Vishal Koparde, Xianyu Zhang, Christine Alewine. Soluble mesothelin acts as a signaling molecule [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 93.

Abstract 1085: PRO1184, a novel folate receptor alpha-directed antibody-drug conjugate, demonstrates robust anti-tumor activity in mouse carcinoma models

Abstract PRO1184 is an antibody-drug conjugate (ADC) directed toward folate receptor alpha (FOLR1), a glycosyl phosphatidylinositol anchored membrane protein. FOLR1 is overexpressed in many cancers with unmet medical need, including ovarian, lung, and breast cancers, while normal tissue expression is limited. PRO1184 is comprised of a 1) human monoclonal antibody that selectively binds to FOLR1, 2) a cleavable, hydrophilic linker, and 3) exatecan, a topoisomerase 1 inhibitor. Upon binding to FOLR1 on the surface of malignant cells, PRO1184 is internalized and exatecan released through enzymatic cleavage of the linker. Exatecan blocks the ligation step of the cell cycle and generates DNA single- and double-strand breaks, which leads to cell death. The mechanism of action and clinical potential of PRO1184 was tested in a series of studies. PRO1184 bound selectively and specifically to FOLR1 with nM affinity. PRO1184 was efficiently internalized and demonstrated cytotoxicity against multiple cell lines, in vitro. In mouse carcinoma models, PRO1184 demonstrated robust anti-tumor activity across multiple tumor types that represent ovarian, non-small cell lung, and breast cancer. In addition, PRO1184 was more potent with greater tumor growth inhibition than that of a DM4-conjugated ADC. PRO1184 was tolerated at the 60 mg/kg dose level in cynomolgus monkeys and the safety profile was generally more favorable than that for a DXd-based ADC. The pharmacokinetics (PK) of PRO1184 were similar to that of the unconjugated parent antibody in rats. PRO1184 has the potential for a meaningful therapeutic window to provide an appropriate benefit to risk profile for patients with FOLR1-expressing cancers. Citation Format: Baiteng Zhao, Lei Wang, Haidong Liu, Suping Huang, Xiao Shang, Tae Han. PRO1184, a novel folate receptor alpha-directed antibody-drug conjugate, demonstrates robust anti-tumor activity in mouse carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1085.

Influenza Virus-like Particle-Based Hybrid Vaccine Containing RBD Induces Immunity against Influenza and SARS-CoV-2 Viruses.

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

Thy-1-Integrin Interactions in cis and Trans Mediate Distinctive Signaling.

Thy-1 is a cell surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein that bears a broad mosaic of biological roles across various cell types. Thy-1 displays strong physiological and pathological implications in development, cancer, immunity, and tissue fibrosis. Quite uniquely, Thy-1 is capable of mediating integrin-related signaling through direct trans- and cis-interaction with integrins. Both interaction types have shown distinctive roles, even when interacting with the same type of integrin, where binding in trans or in cis often yields divergent signaling events. In this review, we will revisit recent progress and discoveries of Thy-1–integrin interactions in trans and in cis, highlight their pathophysiological consequences and explore other potential binding partners of Thy-1 within the integrin regulation/signaling paradigm.

Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of β-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse β-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non–GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

Establishment of mouse model of inherited PIGO deficiency and therapeutic potential of AAV-based gene therapy

Inherited glycosylphosphatidylinositol (GPI) deficiency (IGD) is caused by mutations in GPI biosynthesis genes. The mechanisms of its systemic, especially neurological, symptoms are not clarified and fundamental therapy has not been established. Here, we report establishment of mouse models of IGD caused by PIGO mutations as well as development of effective gene therapy. As the clinical manifestations of IGD are systemic and lifelong lasting, we treated the mice with adeno-associated virus for homology-independent knock-in as well as extra-chromosomal expression of Pigo cDNA. Significant amelioration of neuronal phenotypes and growth defect was achieved, opening a new avenue for curing IGDs.

Nutrition interventions in congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are a group of more than 160 inborn errors of metabolism affecting multiple pathways of protein and lipid glycosylation. Patients present with a wide range of symptoms and therapies are only available for very few subtypes. Specific nutritional treatment options for certain CDG types include oral supplementation of monosaccharide sugars, manganese, uridine, or pyridoxine. Additional management includes specific diets (i.e., complex carbohydrate or ketogenic diet), iron supplementation, and albumin infusions. We review the dietary management in CDG with a focus on two subgroups: N-linked glycosylation defects and GPI-anchor disorders.

The role of glycosylphosphatidylinositol (gpi) anchored proteins in Cryptococcusneoformans

It is becoming increasingly obvious that glycophosphatidylinositol (GPI)-anchored proteins (GAPs) play a prominent role in fungi, a full understanding of GAPs is however lacking especially for the human opportunistic fungus Cryptococcus neoformans. Using online GPI prediction tools, GAPs were identified and subsequently a mutant library for these GAP-encoding genes was developed and a publicly available knock out (KO) mutant library was used. In total, 41 overexpression and 34 KO mutants, representing 47 unique genes, were analyzed. From the analysis of the two libraries, two main gene candidates, a mannoprotein 88 (MP88) (CNAG_00776) and an uncharacterized protein (CNAG_00137) were further investigated by constructing additional independent mutant strains. The CNAG_00776 mutant showed an impaired growth upon plasma membrane stress and significant decreased phagocytosis. The CNAG_00137 mutant showed impaired growth during cell wall stress or increased temperature and significant decreased phagocytosis. By performing a large genetic screen of GAPs in the genome of the human fungal pathogen C. neoformans, we identified two candidate GAP genes involved in C. neoformans/host interaction and stress response. Further research into these two genes could potentially result in new targets for antfungals, treatment strategies or vaccines to manage C. neoformans disease.

Clostridium septicum α-toxin activates the NLRP3 inflammasome by engaging GPI-anchored proteins.

Clostridium species are a group of Gram-positive bacteria that cause diseases in humans, such as food poisoning, botulism, and tetanus. Here, we analyzed 10 different Clostridium species and identified that Clostridium septicum, a pathogen that causes sepsis and gas gangrene, activates the mammalian cytosolic inflammasome complex in mice and humans. Mechanistically, we demonstrate that α-toxin secreted by C. septicum binds to glycosylphosphatidylinositol (GPI)-anchored proteins on the host plasma membrane, oligomerizing and forming a membrane pore that is permissive to efflux of magnesium and potassium ions. Efflux of these cytosolic ions triggers the activation of the innate immune sensor NLRP3, inducing activation of caspase-1 and gasdermin D, secretion of the proinflammatory cytokines interleukin-1β and interleukin-18, pyroptosis, and plasma membrane rupture via ninjurin-1. Furthermore, α-toxin of C. septicum induces rapid inflammasome-mediated lethality in mice and pharmacological inhibition of the NLRP3 inflammasome using MCC950 prevents C. septicum-induced lethality. Overall, our results reveal that cytosolic innate sensing of α-toxin is central to the recognition of C. septicum infection and that therapeutic blockade of the inflammasome pathway may prevent sepsis and death caused by toxin-producing pathogens.

Ethanolamine-phosphate on the second mannose is a preferential bridge for some GPI-anchored proteins.

Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor many proteins (GPI-APs) on the cell surface. The core glycan of GPI precursor has three mannoses, which in mammals, are all modified by ethanolamine-phosphate (EthN-P). It is postulated that EthN-P on the third mannose (EthN-P-Man3) is the bridge between GPI and the protein and the second (EthN-P-Man2) is removed after GPI-protein attachment. However, EthN-P-Man2 may not be always transient, as mutations of PIGG, the enzyme that transfers EthN-P to Man2, result in inherited GPI deficiencies (IGDs), characterized by neuronal dysfunctions. Here, we show that EthN-P on Man2 is the preferential bridge in some GPI-APs, among them, the Ect-5'-nucleotidase and Netrin G2. We find that CD59, a GPI-AP, is attached via EthN-P-Man2 both in PIGB-knockout cells, in which GPI lacks Man3, and with a small fraction in wild-type cells. Our findings modify the current view of GPI anchoring and provide a mechanistic basis for IGDs caused by PIGG mutations.

Novel top-down high-resolution mass spectrometry-based metabolomics and lipidomics reveal molecular change mechanism in A2 milk after CSN2 gene mutation

The effects of milk containing only native A2 beta casein (A2 milk) on amelioration of gut tolerance are widely studied by clinical in vivo trials. However, the molecular change mechanism after CSN2 gene mutation and other potential health benefits in A2 milk remain unknown. The glycerophospholipid metabolism, sphingolipid metabolism, glycerolipid metabolism, phosphatidylinositol signaling system and glycosylphosphatidylinositol (GPI)-anchor biosynthesis were considered as significant metabolic pathways intimately related with the mutation of CSN2 gene by integrating metabolomics and lipidomics and the corresponding molecular mechanism was summarized. The contents of lactose, triacylglycerol and most polar lipids significantly decreased after CSN2 gene mutation by utilizing UHPLC-Q-Orbitrap HRMS (LOQs, 0.0004–0.0215 μg/mL) combined with multivariate analysis, furthermore, the essential fatty acids α-linolenic acid (1015.22 to 543.14 μg/mL) and arachidonic acid were significantly down regulated although eicosapentaenoic acid was relatively upregulated, which provided a novel perspective to understand the nutritional value and health benefits of A2 milk.

A genetic screen identifies a protective type III interferon response to Cryptosporidium that requires TLR3 dependent recognition.

Cryptosporidium is a leading cause of severe diarrhea and diarrheal-related death in children worldwide. As an obligate intracellular parasite, Cryptosporidium relies on intestinal epithelial cells to provide a niche for its growth and survival, but little is known about the contributions that the infected cell makes to this relationship. Here we conducted a genome wide CRISPR/Cas9 knockout screen to discover host genes that influence Cryptosporidium parvum infection and/or host cell survival. Gene enrichment analysis indicated that the host interferon response, glycosaminoglycan (GAG) and glycosylphosphatidylinositol (GPI) anchor biosynthesis are important determinants of susceptibility to C. parvum infection and impact on the viability of host cells in the context of parasite infection. Several of these pathways are linked to parasite attachment and invasion and C-type lectins on the surface of the parasite. Evaluation of transcript and protein induction of innate interferons revealed a pronounced type III interferon response to Cryptosporidium in human cells as well as in mice. Treatment of mice with IFNλ reduced infection burden and protected immunocompromised mice from severe outcomes including death, with effects that required STAT1 signaling in the enterocyte. Initiation of this type III interferon response was dependent on sustained intracellular growth and mediated by the pattern recognition receptor TLR3. We conclude that host cell intrinsic recognition of Cryptosporidium results in IFNλ production critical to early protection against this infection.

The distribution and function of GDE2, a regulator of spinal motor neuron survival, are disrupted in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the viability of upper and lower motor neurons. Current options for treatment are limited, necessitating deeper understanding of the mechanisms underlying ALS pathogenesis. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane protein that acts on the cell surface to cleave the glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane. GDE2 is required for the survival of spinal motor neurons but whether GDE2 neuroprotective activity is disrupted in ALS is not known. We utilized a combination of mouse models and patient post-mortem samples to evaluate GDE2 functionality in ALS. Haplogenetic reduction of GDE2 exacerbated motor neuron degeneration and loss in SOD1G93A mice but not in control SOD1WT transgenic animals, indicating that GDE2 neuroprotective function is diminished in the context of SOD1G93A. In tissue samples from patients with ALS, total levels of GDE2 protein were equivalent to healthy controls; however, membrane levels of GDE2 were substantially reduced. Indeed, GDE2 was found to aberrantly accumulate in intracellular compartments of ALS motor cortex, consistent with a disruption of GDE2 function at the cell surface. Supporting the impairment of GDE2 activity in ALS, tandem-mass-tag mass spectrometry revealed a pronounced reduction of GPI-anchored proteins released into the CSF of patients with ALS compared with control patients. Taken together, this study provides cellular and biochemical evidence that GDE2 distribution and activity is disrupted in ALS, supporting the notion that the failure of GDE2-dependent neuroprotective pathways contributes to neurodegeneration and motor neuron loss in disease. These observations highlight the dysregulation of GPI-anchored protein pathways as candidate mediators of disease onset and progression and accordingly, provide new insight into the mechanisms underlying ALS pathogenesis.

Novel Polymorphisms and Genetic Characteristics of the Shadow of Prion Protein Gene (SPRN) in Cats, Hosts of Feline Spongiform Encephalopathy.

Prion diseases are transmissible spongiform encephalopathies (TSEs) caused by pathogenic prion protein (PrPSc) originating from normal prion protein (PrPC) and have been reported in several types of livestock and pets. Recent studies have reported that the shadow of prion protein (Sho) encoded by the shadow of prion protein gene (SPRN) interacts with prion protein (PrP) and accelerates prion diseases. In addition, genetic polymorphisms in the SPRN gene are related to susceptibility to prion diseases. However, genetic polymorphisms in the feline SPRN gene and structural characteristics of the Sho have not been investigated in cats, a major host of feline spongiform encephalopathy (FSE). We performed amplicon sequencing to identify feline SPRN polymorphisms in the 623 bp encompassing the open reading frame (ORF) and a small part of the 3′ untranslated region (UTR) of the SPRN gene. We analyzed the impact of feline SPRN polymorphisms on the secondary structure of SPRN mRNA using RNAsnp. In addition, to find feline-specific amino acids, we carried out multiple sequence alignments using ClustalW. Furthermore, we analyzed the N-terminal signal peptide and glycosylphosphatidylinositol (GPI)-anchor using SignalP and PredGPI, respectively. We identified three novel SNPs in the feline SPRN gene and did not find strong linkage disequilibrium (LD) among the three SNPs. We found four major haplotypes of the SPRN polymorphisms. Strong LD was not observed between PRNP and SPRN polymorphisms. In addition, we found alterations in the secondary structure and minimum free energy of the mRNA according to the haplotypes in the SPRN polymorphisms. Furthermore, we found four feline-specific amino acids in the feline Sho using multiple sequence alignments among several species. Lastly, the N-terminal signal sequence and cutting site of the Sho protein of cats showed similarity with those of other species. However, the feline Sho protein exhibited the shortest signal sequence and a unique amino acid in the omega-site of the GPI anchor. To the best of our knowledge, this is the first report on genetic polymorphisms of the feline SPRN gene.

Establishment and Application of an Indirect Enzyme-Linked Immunosorbent Assay for Measuring GPI-Anchored Protein 52 (P52) Antibodies in Babesia gibsoni-Infected Dogs.

Simple SummaryIn this study, a novel BgGPI52-WH antigen was identified and evaluated as an immunodiagnostic candidate. An indirect ELISA was established based on the BgGPI52-WH antigen. The results showed that the iELISA had good sensitivity, specificity and reproducibility, and the signal could be detected as early as the sixth day after infection. Clinical samples were tested using the established method, and 11.41% of the samples were positive. The results suggested that BgGPI52-WH is a good immunodiagnostic marker and the iELISA is a practical method for early diagnosis.Babesia gibsoni is a malaria-like protozoan that parasitizes the red blood cells of canids to cause babesiosis. Due to its high expression and essential function in the survival of parasites, the Glycosylphosphatidylinositol (GPI) anchor protein family is considered an excellent immunodiagnostic marker. Herein, we identified a novel GPI-anchored protein named as BgGPI52-WH with a size of 52 kDa; the recombinant BgGPI52-WH with high antigenicity and immunogenicity was used as a diagnostic antigen to establish a new iELISA method. The iELISA had a sensitivity of 1:400, and no cross-reaction with other apicomplexan parasites occurred. We further demonstrated that the degree of variation was less than 10% using the same samples from the same or different batches of an enzyme-labeled strip. It was found that the method was able to detect early infection (6 days after infection) in the sera of the B. gibsoni-infected experimental dogs in which antibody response to rBgGPI52-WH was evaluated. Clinical sera from pet hospitals were further tested, and the average positive rate was about 11.41% (17/149). The results indicate that BgGPI52-WH is a reliable diagnostic antigen, and the new iELISA could be used as a practical method for the early diagnosis of B. gibsoni.