Glycosylphosphatidylinositol Research Articles

Proteolytic cleavage of the TGFβ co-receptor CD109 changes its conformation, resulting in protease inhibition via activation of its thiol ester, and dissociation from the cell membrane.

The glycosylphosphatidylinositol (GPI)-anchored protein cluster of differentiation 109 (CD109) is expressed on many human cell types and modulates the transforming growth factor β (TGF-β) signaling network. CD109 belongs to the alpha-macroglobulin family of proteins, known for their protease-triggered conformational changes. However, the effect of proteolysis on CD109 and its conformation are unknown. Here, we investigated the interactions of CD109 with proteases. We found that a diverse selection of proteases cleaved peptide bonds within the predicted bait region of CD109, inducing a conformational change that activated the thiol ester of CD109. We show CD109 was able to conjugate proteases with this thiol ester and decrease their activity toward protein substrates, demonstrating that CD109 is a protease inhibitor. We additionally found that CD109 has a unique mechanism whereby its GPI-anchored macroglobulin 8 (MG8) domain dissociates during its conformational change, allowing proteases to release CD109 from the cell surface by a precise mechanism and not unspecific shedding. We conclude that proteolysis of the CD109 bait region affects both its structure and location, and that interactions between CD109 and proteases may be important to understanding its functions, for example, as a TGF-β co-receptor.

Abstract LB330: CWH43 as a prognostic marker and therapeutic target in colorectal cancer

Abstract Colorectal cancer (CRC) remains a significant global health issue, particularly challenging due to the poor prognosis of its metastatic form. This highlights the critical need for novel and effective therapeutic targets to manage disease progression. A key focus of our research is the Cell Wall Biogenesis 43 C-Terminal Homolog (CWH43), which plays an essential role in GPI anchor biosynthesis and lipid remodeling, crucial for attaching membrane proteins to cell membranes. Our study aimed to clarify the function of CWH43 in CRC progression and evaluate its potential as a therapeutic target. Through an integrated bioinformatics analysis using public cancer databases, coupled with experimental validations in CRC cell models, we noted a conspicuous decrease in CWH43 mRNA levels in CRC tissues compared to normal tissue. This decrease in CWH43 expression correlated with poorer prognoses in CRC patients. In our experimental findings, we discovered that suppressing CWH43 led to increased proliferation, migration, and invasiveness of CRC cells, whereas its overexpression inhibited these processes. Notably, we observed elevated levels of CWH43 in CRC patients who showed a lower response to chemotherapy. Particularly in advanced-stage CRC patients (T4 and N2), high CWH43 expression correlated with reduced overall survival and shorter progression-free intervals, suggesting a potential association with chemotherapy resistance. In our cellular experiments, overexpression of CWH43 in CRC cells resulted in decreased sensitivity to chemotherapy drugs like CPT11, 5-FU, and oxaliplatin, which was consistent with a reduction in the expression of apoptosis-related markers. Additionally, there was a notable negative correlation between CWH43 expression and radiation sensitivity. Our Gene Set Enrichment Analysis (GSEA) revealed a significant connection between CWH43 and pathways involved in oxidative phosphorylation and fatty acid metabolism, indicating its role in regulating ferroptosis. We also demonstrated that overexpressing CWH43 in CRC cells increases the levels of multiple ferroptosis suppressors. Importantly, the use of ferroptosis inducers appeared to counteract the chemotherapy resistance influenced by CWH43. Based on these findings, we propose that CWH43 plays a role in inhibiting ferroptosis, contributing to therapeutic resistance in CRC. Therefore, targeting CWH43 could be a promising strategy for developing ferroptosis-inducing therapies, offering a new avenue for the treatment of CRC, especially in cases resistant to traditional therapies. Citation Format: Cheng-Chin Lee, Yu-Jia Chang, Chien-Yu Huang. CWH43 as a prognostic marker and therapeutic target in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB330.

Loss of GDE2 leads to complex behavioral changes including memory impairment

BackgroundAlzheimer’s disease (AD) and amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) are debilitating neurodegenerative diseases for which there are currently no cures. Familial cases with known genetic causes make up less than 10% of these diseases, and little is known about the underlying mechanisms that contribute to sporadic disease. Accordingly, it is important to expand investigations into possible pathways that may contribute to disease pathophysiology. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is a membrane-bound enzyme that acts at the cell surface to cleave the glycosylphosphatidylinositol (GPI)-anchor that tethers distinct proteins to the membrane. GDE2 abnormally accumulates in intracellular compartments in the brain of patients with AD, ALS, and ALS/FTD, indicative of GDE2 dysfunction. Mice lacking GDE2 (Gde2KO) show neurodegenerative changes such as neuronal loss, reduced synaptic proteins and synapse loss, and increased Aβ deposition, raising the possibility that GDE2 disruption in disease might contribute to disease pathophysiology. However, the effect of GDE2 loss on behavioral function and learning/memory has not been characterized.ResultsHere, we show that GDE2 is expressed throughout the adult mouse brain in areas including the cortex, hippocampus, habenula, thalamus, and amygdala. Gde2KO and WT mice were tested in a set of behavioral tasks between 7 and 16 months of age. Compared to WT, Gde2KO mice display moderate hyperactivity that becomes more pronounced with age across a variety of behavioral tests assessing novelty-induced exploratory activity. Additionally, Gde2KO mice show reduced startle response, with females showing additional defects in prepulse inhibition. No changes in anxiety-associated behaviors were found, but Gde2KOs show reduced sociability. Notably, aged Gde2KO mice demonstrate impaired short/long-term spatial memory and cued fear memory/secondary contextual fear acquisition.ConclusionsTaken together, these observations suggest that loss of GDE2 leads to behavioral deficits, some of which are seen in neurodegenerative disease models, implying that loss of GDE2 may be an important contributor to phenotypes associated with neurodegeneration.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

Identification of a novel protein localized to the crystalloid of the Plasmodium ookinete

Reducing Plasmodium parasite transmission via the mosquito vector is a promising strategy for malaria control and elimination in endemic regions. In the mosquito midgut after the ingestion of an infected blood meal, malaria parasite gametes egress from erythrocytes and fertilize to develop into motile ookinetes that traverse midgut epithelial cells and transform into oocysts adjacent the basal lamina. Plasmodium ookinetes and young oocysts possess a unique organelle called the crystalloid; which has a honeycomb-like matrix structure and is indicated to be involved in sporozoite formation and maturation. In this study, we identified a novel crystalloid protein, PY17X_1113800, that is exclusively expressed in developing ookinetes. The protein possesses a signal peptide sequence, but lacks a transmembrane domain or GPI anchor signal sequence, as well as predicted adhesive domains which are characterisitic of many crystalloid proteins. The protein is highly conserved across the phylum Apicomplexa and within the greater clade Alveolata, such as Vitrella and the ciliates Paramecium and Tetrahymena, but is absent in cryptosporidia.

Abstract 3637: Ultrasensitive assays for combined detection of CEA+ extracellular vesicles and soluble CEA

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and is the second leading cause of cancer death in the United States. Carcinoembryonic antigen (CEA) is a biomarker commonly used in the management of CRC. It is typically present at low levels in the blood of healthy adults but is elevated in CRC samples and some other cancers. CEA is most commonly used to monitor treatment efficacy and as a critical part of post-treatment surveillance for residual disease, recurrence, and/or progression/metastasis. The CRC recurrence rate averages >25%, and liver metastasis occurs in 25-30% of cases. Improved CEA assays could enable earlier determination of treatment failure, detection of lower levels of minimal residual disease (MRD) after treatment, or earlier detection of recurrence. CEA is a GPI-linked membrane protein secreted from CRC cells mainly by enzymatic cleavage of its GPI anchor. However, we have identified alternative secretion of CEA in association with extracellular vesicles (EVs). We hypothesized that the soluble CEA produced by canonical shedding and the alternatively secreted CEA may provide distinct information about the tumor or its microenvironment and serve as independent but complementary biomarkers of CRC. We developed ultrasensitive, multi-marker electrochemiluminescence (ECL) immunoassays for measuring intact EVs presenting CEA, CD73, or both in human plasma. We observed that plasma levels of CEA+EVs and CD73+EVs were elevated in some late-stage CRC samples compared to controls. CEA and CD73 double-positive EV (CEA+CD73+EVs) levels were also elevated in the same samples, exhibiting a 3-fold improved separation of these samples with CRC from controls, compared to detection of CEA+EVs. Additionally, approximately 25% of stage IV CRC samples had elevated CEA+EV and CEA+CD73+EV levels while exhibiting low soluble CEA. We confirmed this result in an independent sample set that included samples across all CRC stages, and also identified a few earlier-stage samples that exhibited high CEA+EVs and CEA+CD73+EVs without elevated soluble CEA. Since CEA+CD73+EVs and soluble CEA assays identified different subsets of samples, we combined the two assays into a single classifier for late stage CRC and controls, yielding improved receiver operator characteristic (ROC) with area under the curve (AUC) of 0.975 versus 0.927 for the standard soluble CEA assay or 0.788 for CEA+CD73+EVs alone. These results suggest that assaying EVs presenting multiple surface markers can increase specificity for the detection of tumor-derived EVs relative to a single marker and that assaying both soluble and EV-associated forms of CEA in plasma may improve the value of CEA measurement in CRC. Citation Format: Lucie Hebert, Misk Al-Ameen, Talis Spriggs, Collin Nelson, Evan A. Gizzie, Jeffrey L. Franklin, Robert J. Coffey, David A. Routenberg, Jacob N. Wohlstadter. Ultrasensitive assays for combined detection of CEA+ extracellular vesicles and soluble CEA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3637.

Abstract 3124: MRG006A, a novel glypican-3-targeting antibody-drug conjugate, demonstrated potent anti-tumor activity and good safety profile in preclinical studies

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most challenging cancers with high unmet medical need. Glypican-3 (GPC3) is an oncofetal glycoprotein anchored to the cell surface via GPI (glycosylphosphatidylinositol) and frequently over-expressed in HCC with limited expression in normal adult tissues, which makes it an appealing antibody-drug conjugate (ADC) target for HCC. MRG006A is a potentially first-in-class GPC3-targeting ADC composed of a novel humanized IgG1 antibody conjugated to a proprietary linker-payload platform utilizing a potent topoisomerase 1 inhibitor via a peptide-based cleavable linker. Materials and Methods: As a GPC3-targeting ADC intended for clinical therapy of GPC3-expressing cancers, the mechanism of action and therapeutic potential of MRG006A were investigated in a series of preclinical studies, including (1) MRG006A and its antibody moiety binding to human GPC3 expressed on cells assessed by flow cytometry; (2) binding affinity to human and cynomolgus monkey GPC3 measured by SPR (surface plasmon resonance); (3) internalization in Huh7 cells assessed by pHAb Reactive Dye and confocal microscopy; (4) in vitro cytotoxicity against a panel of HCC cell lines (Huh7, HepG2, Hep3B and GPC3-negative SK-HEP-1); (5) anti-tumor activity of MRG006A evaluated in Huh7 and HepG2 cell line-derived xenograft (CDX) as well as a panel of HCC patient-derived xenograft (PDX) mouse models with a different expression level of GPC3; (6) plasma stability of MRG006A evaluated in human, cynomolgus monkey, rat and mouse plasma; and (7) an exploratory pharmacokinetic (PK) and toxicology study of MRG006A in cynomolgus monkey. Results: The GPC3-targeting ADC, MRG006A, and its antibody moiety exhibited superior binding activity to GPC3-expressing cancer cell lines than a clinical antibody benchmark and similar nanomolar binding affinity to both human and cynomolgus monkey GPC3, which makes cynomolgus monkey suitable for toxicological studies. Rapid internalization of MRG006A antibody was observed in GPC3-expressing Huh7 cells, and MRG006A maintained comparable internalization capability to the naked antibody. More importantly, MRG006A exhibited potent GPC3-dependent cytotoxic activity in a panel of HCC cells expressing varying levels of GPC3. Administrations of MRG006A resulted in a robust and dose-dependent tumor growth inhibition of multiple CDX models and HCC PDX models. Remarkable stability of MRG006A was shown in the plasma of various species tested. MRG006A demonstrated a favorable PK and safety profile with good tolerability in the exploratory toxicology study. Conclusion: Overall, the preclinical study results suggest that MRG006A is a feasible ADC drug candidate for treating GPC3-expressing cancers in clinical studies. MRG006A was aimed to submit IND in China and the U.S in 2024. Citation Format: Yanchun Wang, Hongfeng Li, Hao Shen, Wenchao Liu, Shoujia Liu, Kequan Yin, Haili Xu, Xueyuan Cui, Wei Li, Wei Liu, Xiangyu Wu, Liu Yang, Tian Ma, Zhongrun Zhao, Jun Wang, Feifei Cui, Lei Fang, Minmin Qin, Chaohong Hu. MRG006A, a novel glypican-3-targeting antibody-drug conjugate, demonstrated potent anti-tumor activity and good safety profile in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3124.

In silico prediction of CD8+ and CD4+ T cell epitopes in Leishmania major proteome: Using immunoinformatics

The leishmaniases are NDTs (neglected tropical diseases) that affect people all over the world. They are brought on by protozoans from the genus Leishmania and disseminated by phlebotomine flies that are afflicted with the disease. The best option to manage and lower the incidence of these diseases has been thought by the creation of a safe and effective vaccination. This research used an in silico based mining approach to look for high potential epitopes that might bind to MHC Class I and MHC Class II molecules (mainly; HLA-A*02:01 & HLA-DRB1*03:01) from human population in order to promote vaccine development. Based on the presence of signal peptides, GPI anchors, antigenicity predictions, and a subtractive proteomic technique, we have screened 17 putative antigenic proteins from the 8083 total proteins of L. major. After that thorough immunogenic epitope prediction were done using IEDB-AR tools. We isolated five immunogenic epitopes (three 9-mer & two 15-mer) from five antigenic proteins through docking and MD simulation analysis. Finally, these five anticipated epitopes, viz., TLPEIPVNV, ELMAPVFGL, TLAAAVALL, NSINIRLDGVTSAGF and NVPLVVDASSLFRVA have considerably stronger binding potential with their respective alleles and may trigger immunological responses. The goal of this work was to identify MHC restricted epitopes for CD8+ and CD4+ T cells activation using immunoinformatics in order to identify potential vaccine candidates against L. major parasites.

Proteomic Analysis of Cell Wall Proteins with Various Linkages in Fusarium graminearum.

The fungal cell wall, primarily comprising a glucan-chitin matrix and cell wall proteins (CWPs), serves as a key mediator for fungal interactions with the environment and plays a pivotal role in virulence. In this study, we employed a comprehensive proteomics approach to analyze the CWPs in the plant pathogenic fungus Fusarium graminearum. Our methodology successfully extracted and identified 1373 CWPs, highlighting their complex linkages, including noncovalent bonds, disulfide bridges, alkali-sensitive linkages, and glycosylphosphatidylinositol (GPI) anchors. A significant subset of these proteins, enriched in Gene Ontology terms, suggest multifunctional roles of CWPs. Through the integration of transcriptomic and proteomic data, we observed differential expression patterns of CWPs across developmental stages. Specifically, we focused on two genes, Fca7 and Cpd1, which were upregulated in planta, and confirmed their localization predominantly outside the plasma membrane, primarily in the cell wall and periplasmic space. The disruption of FCA7 reduced virulence on wheat, aligning with previous findings and underscoring its significance. Overall, our findings offer a comprehensive proteomic profile of CWPs in F. graminearum, laying the groundwork for a deeper understanding of their roles in the development and interactions with host plants.

The CD177 c.1291A Allele Leads to a Loss of Membrane Expression and Mimics a CD177-Null Phenotype.

CD177 is a glycosyl phosphatidyl inositol (GPI)-linked, neutrophil-specific glycoprotein that in 3-5% of normal individuals is absent from all neutrophils. The molecular mechanism behind the absence of CD177 has not been unravelled completely. Here, we analyse the impact of the recently described CD177 c.1291G>A variant on CD177 expression. Recombinant CD177 c.1291G>A was expressed in HEK293F cells and its expression on the cell surface, inside the cell, and in the culture supernatant was investigated. The CD177 c.1291G>A protein was characterised serologically and its interaction with proteinase 3 (PR3) was demonstrated by confocal laser scanning microscopy. Our experiments show that CD177 c.1291G>A does not interfere with CD177 protein biosynthesis but affects the membrane expression of CD177, leading to very low copy numbers of the protein on the cellular surface. The mutation does not interfere with the ability of the protein to bind PR3 or human polyclonal antibodies against wild-type CD177. Carriers of the c.1291G>A allele are supposed to be phenotyped as CD177-negative, but the protein is present in soluble form. The presence of CD177 c.1291A leads to the production of an unstable CD177 protein and an apparent "CD177-null" phenotype.

PIGN c.776T>C (p.Phe259Ser) variant present in trans with a pathogenic variant for PIGN-congenital disorder of glycosylation: Bella-Noah syndrome

Glycosylation is the most common protein and lipid post-translational modification in humans. Congenital disorders of glycosylation (CDG) are characterized by both genetic and clinical heterogeneity, presenting multisystemic manifestations, and in most cases are autosomal recessive in inheritance. The PIGN gene is responsible for the addition of phosphoethanolamine to the first mannose in the glycosylphosphatidylinositol (GPI)-anchor biosynthesis pathway, a highly conserved process that enables proteins to bind to the cell surface membrane. Here, we report a family with two siblings pediatric cases with the exact same compound heterozygous variants in PIGN. The (c.776T > C) variant of uncertain significance (VUS) together with a known pathogenic variant (c.932T > G), resulting in clinical features compatible with PIGN-related conditions, more specific the CDG. This is the first time that PIGN variant c.776T > C is reported in literature in individuals with PIGN-congenital disorder of glycosylation (PIGN-CDG), and the current submission in ClinVar by Invitae® is specifically of our case. Detailed clinical information and molecular analyses are presented. Here, we show for the first time two affected siblings with one pathogenic variant (c.932T > G) and the c.776T > C VUS in trans. In honor of the family, we propose the name Bella-Noah Syndrome for disorder.

Abstract 1046 Identification of the GPI Anchor Sidechain Modifying Enzymes in Toxoplasma gondii and Their Role in Virulence

Abstract 1046 Identification of the GPI Anchor Sidechain Modifying Enzymes in Toxoplasma gondii and Their Role in Virulence

Checkpoint CD24 function on tumor and immunotherapy.

CD24 is a protein found on the surface of cells that plays a crucial role in the proliferation, invasion, and spread of cancer cells. It adheres to cell membranes through glycosylphosphatidylinositol (GPI) and is associated with the prognosis and survival rate of cancer patients. CD24 interacts with the inhibitory receptor Siglec-10 that is present on immune cells like natural killer cells and macrophages, leading to the inhibition of natural killer cell cytotoxicity and macrophage-mediated phagocytosis. This interaction helps tumor cells escape immune detection and attack. Although the use of CD24 as a immune checkpoint receptor target for cancer immunotherapy is still in its early stages, clinical trials have shown promising results. Monoclonal antibodies targeting CD24 have been found to be well-tolerated and safe. Other preclinical studies are exploring the use of chimeric antigen receptor (CAR) T cells, antibody-drug conjugates, and gene therapy to target CD24 and enhance the immune response against tumors. In summary, this review focuses on the role of CD24 in the immune system and provides evidence for CD24 as a promising immune checkpoint for cancer immunotherapy.

Effect of high NEFA concentration on lipid metabolism disorders in hepatocytes based on lipidomics.

Introduction: High concentrations of nonesterified fatty acids (NEFA) is the key of characteristic of fatty liver in dairy cows. Therefore, the aim of this study was to investigate the effect of high concentration of NEFA on lipid metabolism in hepatocytes through the lipidomic approach and molecular biology techniques. Methods: Stimulate AML-12 cells with different concentrations of NEFA, observe the cellular lipid accumulation, and select 0.6mM NEFA stimulation concentration for subsequent experiments. Collect cells for lipidomics analysis. Results: High concentration of NEFA (0.6-2.4mM) significantly reduced the cell viability in a concentration-dependent manner, indicating that high concentrations of NEFA have lipotoxicity on hepatocytes. In addition, NEFA promoted triglycerides (TAG) accumulation, increased the mRNA expression of the lipogenic molecules SREBP1c and FASN, and decreased the mRNA expression of lipolytic molecules CPT1A and HSL in hepatocytes. Mechanistically, high concentration of NEFA induced lipid metabolism disorders in hepatocytes by regulating metabolic pathways such as glycerol phospholipid metabolism, glycosyl phosphatidylinositol anchored biosynthesis, triglyceride metabolism, sphingolipid metabolism, and inositol phosphate metabolism. Discussion: High concentration of NEFA is lipotoxic to cells, promoting lipid accumulation. LPE (18:2), LPE (18:3), LPE (18:1) via glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, glycerolipid metabolism, sphingolipid metabolism, and inositol phosphate metabolism, indicating their potential regulation role in the pathogenesis of fatty liver.

Production of CA125 with Tn antigens using a glycosylphosphatidylinositol anchoring system.

Cancer antigen 125 (CA125) is a serum marker associated with ovarian cancer. Despite its widespread use, CA125 levels can also be elevated in benign conditions. Recent reports suggest that detecting serum CA125 that carries the Tn antigen, a truncated O-glycan containing only N-acetylgalactosamine on serine or threonine residues, can improve the specificity of ovarian cancer diagnosis. In this study, we engineered cells to express CA125 with a Tn antigen. To achieve this, we knocked out C1GALT1 and SLC35A1, genes encoding Core1 synthase and a transporter for cytidine-5'-monophospho-sialic acid respectively, in human embryonic kidney 293 (HEK293) cells. In ClGALT1-SLC35A1-knockout (KO) cells, the expression of the Tn antigen showed a significant increase, whereas the expression of the T antigen (galactose-β1,3-N-acetylgalactosamine on serine or threonine residues) was decreased. Due to the inefficient secretion of soluble CA125, we employed a glycosylphosphatidylinositol (GPI) anchoring system. This allowed for the expression of GPI-anchored CA125 on the cell surface of ClGALT1-SLC35A1-KO cells. Cells expressing high levels of GPI-anchored CA125 were then enriched through cell sorting. By knocking out the PGAP2 gene, the GPI-anchored form of CA125 was converted to a secretory form. Through the engineering of O-glycans and the use of a GPI-anchoring system, we successfully produced CA125 with Tn antigen modification.

Extraction-free, immuno-RPA-CRISPR/Cas13a-based one-pot detection of glypican-3 directly from extracellular vesicles

BackgroundGlypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) that binds to the cell membrane via glycosylphosphatidylinositol (GPI). It is not found in healthy adult liver but is overexpressed in human hepatocellular carcinoma (HCC). The protein marker GPC3 on extracellular vesicles (GPC3+ EVs) is also useful for HCC detection. Nevertheless, the absence of practical and dependable quantitative techniques to evaluate EVs proteins prevents their clinical implementation. ResultsHere, using an immuno-recombinase polymerase amplification (immuno-RPA) process and dual amplification of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, we firstly create an extraction-free one-pot immuno-RPA-CRISPR (opiCRISPR) for the direct and extremely sensitive detection of EVs proteins. The EVs protein-targeted detection probe is amplified by RPA to generate a long repetitive sequence containing multiple CRISPR RNA (crRNA) targeting barcodes, and the signal is further amplified by the CRISPR-Cas13a side-chain cleavage activity to generate a fluorescent signal. The results show that circulating extracellular vesicle GPC3 (eGPC3) levels are a reliable marker for GPC3 expression in tumor, opening up new avenues for tumor diagnosis. Significance and noveltyWe created an eGPC3 assay based on the CRISPR-Cas13a system, and successfully study the significance of extracellular vesicle GPC3 markers in hepatocellular carcinoma.

Stable expression of mucin glycoproteins GP40 and GP15 of Cryptosporidium parvum in Toxoplasma gondii

BackgroundCryptosporidium spp. are common protozoa causing diarrhea in humans and animals. There are currently only one FDA-approved drug and no vaccines for cryptosporidiosis, largely due to the limited knowledge of the molecular mechanisms involved in the invasion of the pathogens. Previous studies have shown that GP60, which is cleaved into GP40 and GP15 after expression, is an immunodominant mucin protein involved in the invasion of Cryptosporidium. The protein is highly O-glycosylated, and recombinant proteins expressed in prokaryotic systems are non-functional. Therefore, few studies have investigated the function of GP40 and GP15.MethodsTo obtain recombinant GP40 with correct post-translational modifications, we used CRISPR/Cas9 technology to insert GP40 and GP15 into the UPRT locus of Toxoplasma gondii, allowing heterologous expression of Cryptosporidium proteins. In addition, the Twin-Strep tag was inserted after GP40 for efficient purification of GP40.ResultsWestern blotting and immunofluorescent microscopic analyses both indicated that GP40 and GP15 were stably expressed in T. gondii mutants. GP40 localized not only in the cytoplasm of tachyzoites but also in the parasitophorous vacuoles, while GP15 without the GPI anchor was expressed only in the cytoplasm. In addition, a large amount of recTgGP40 was purified using Strep-TactinXT supported by a visible band of ~ 50 kDa in SDS-PAGE.ConclusionsThe establishment of a robust and efficient heterologous expression system of GP40 in T. gondii represents a novel approach and concept for investigating Cryptosporidium mucins, overcoming the limitations of previous studies that relied on unstable transient transfection, which involved complex steps and high costs.Graphical

Prevalence of Paroxysmal Nocturnal Haemoglobinuria Clone in Aplastic Anaemia: A Single Centre Study

Background: Flow cytometry assay for PNH clone is a compulsory routine test for all aplastic anaemia patients. Objective: To estimate the frequency of PNH clone in aplastic anaemia. Method: Twenty-two known cases of aplastic anaemia patients were enrolled for the study. Flow cytometric quantitation of glycosyl phosphatidyl-inositol (GPI)-anchored proteins deficiency using markers CD14, CD24, CD45, CD59, Fluorescent Aerolysin (FLAER), CD235a (6 markers) were performed. Result: PNH clone was identified in 8 (36.4%) of the study population. Among PNH clone positive patients 7 (87.5%) were suffering from non-severe aplastic anaemia. Conclusion: PNH clone is significantly associated with aplastic anaemia and PNH clone assay should be regularly assayed in aplastic anaemia patients for specific management.

Synthesis of glycosylphosphatidylinositol analogues with an unnatural β-D-glucosamine-(1→6)-myo-inositol motif

Glycosylphosphatidylinositol (GPI) anchors contain a unique α-D-glucosamine-(1→6)-myo-inositol [αGlcN(1,6)Ins] motif in their conserved core structure. To facilitate investigations of the functional roles of this structural motif, two GPI analogues containing unnatural βGlcN(1,6)Ins, instead of αGlcN(1,6)Ins, and an alkyne group at different positions of the GPI core were designed and synthesized. To this end, an orthogonally protected pseudopentasaccharide derivative of GPIs with the βGlcN(1,6)Ins motif was convergently constructed via [3 + 2] glycosylation and used as the common intermediate to prepare both GPI analogues by streamlined synthetic protocols. The pseudopentasaccharide intermediate and developed protocols can be widely applicable to access various GPI analogues with the βGlcN(1,6)Ins motif. The target GPI analogues contain an alkyne, which allows their further modification to introduce various molecular labels via click chemistry, making them useful probes for the study of GPI anchorage. The differences in reactivity and NMR behavior of the two GPI analogues, as well as the differences of these analogues from previously reported GPI derivatives of similar structure containing an αGlcN(1,6)Ins motif, suggest that the 2-O-phosphoethanolamine moiety on mannose-I and the linkage form of GlcN in GPIs can have a decisive impact on the structure, which is likely relevant to biology.

The multifunctional roles of the extracellular matrix in the sessile life of the zygnematophyte Penium margaritaceum: stick, glide and cluster.

Adhesion and consequent adoption of a sessile habit is a common feature of many green algae and was likely a key mechanism in terrestrialization by an ancient zygnematophyte (i.e., the Zygnematophyceae, the group of algae ancestral to land plants). Penium margaritaceum is a unicellular zygnematophyte that exhibits a multistep adhesion mechanism, which leads to the establishment of the sessile habit. Based on microscopic and immunological data, a dense aggregate of fibrils containing arabinogalactan-protein (AGP)-like components covers the cell surface and is responsible for initial adhesion. The AGP-like fibrils are 20 μm in diameter and possess chemical profiles similar to land plant AGPs. The fibrils attach to the inner cell wall layers and are very likely connected to the plasma membrane as glycophosphatidylinositol (GPI) lipid-anchored proteins, as they are susceptible to phospholipase C treatment. The presence of GPI-anchored AGPs in Penium is further supported by the identification of putative Penium homologs of land plant AGP genes responsible for GPI-anchor synthesis. After adhesion, cells secrete a complex heteropolysaccharide-containing extracellular polymeric substance (EPS) that facilitates gliding motility and the formation of cell aggregates. Fucoidan-like polymers, major components of brown algal CWs, are a major constituent of both the EPS and the adhesive layer of the CW and their role in the adhesion process is still to be examined.