GPI-anchored Proteins Research Articles

Insights from Trajectories of PIGA-Mutated Clones in Paroxysmal Nocturnal Hemoglobinuria

Background: Patients with paroxysmal nocturnal hemoglobinuria (PNH) exhibit clonal expansion of hematopoietic cells deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs). Mutations in PIGA gene, involved in the biosynthesis of GPI-APs, result in the loss of GPI-APs from hematopoietic cells. However, PIGA mutations alone are insufficient to drive this clonal expansion. The pathogenesis of clonal expansion in PNH remains elusive, while several hypotheses or factors have been proposed, including immune attacks with concomitant aplastic anemia (AA) or additional mutations offering intrinsic growth advantage. Recently high-sensitivity flow cytometry and next-generation sequencing have allowed us to evaluate the clonal dynamics and architecture more precisely. The method has the potential to clarify the trajectories of PIGA mutated clones before the significant clonal expansion or the diagnosis, thereby to gain insight into the pathogenesis of the clonal expansion in PNH. Methods: To estimate the trajectories of PIGA-mutated clones, we constructed phylogenetic trees using whole genome sequencing (WGS) data of single cell-derived colonies of bone marrow samples from two PNH patients (53 colonies for Case 1 and 75 for Case 2). We used the targeted-sequencing and exome sequencing to detect driver mutations after isolating GPI-deficient and GPI-positive cells using flow cytometry. Results: Case 1 was diagnosed with AA at the age of 28. He developed hemoglobinuria at the age of 40 and was diagnosed with PNH at the age of 45. Bone marrow and peripheral blood samples were collected when he was 47 years old. The PNH clone sizes in granulocytes and red blood cells were 88.7% and 46.1%, respectively. Targeted-sequencing revealed six PIGA mutations in GPI-deficient cells and three BCOR mutations in GPI-positive cells. Case 2 was diagnosed with AA at the age of 33 and with PNH at the age of 34. Bone marrow and peripheral blood samples were collected when she was 59 years old. The PNH clone sizes in granulocytes and red blood cells were 98.6% and 96.3%, respectively. We previously identified chromosomal abnormalities in chr12, with the breakpoint located in an HMGA2 gene (Inoue et al. Blood. 2006). Targeted-sequencing detected no other driver mutations than single PIGA mutation and the HMGA2 mutation.In Case 1, Phylogenetic tree analysis constructed with WGS of single cell-derived colonies revealed multiple PIGA-mutated and BCOR-mutated clones that expanded independently of each other. All BCOR-mutated clones were identified in the GPI-positive population. In Case 2, all PIGA-mutated clones harbored the HMGA2 mutation, while no GPI-positive populations acquired the HMGA2 mutation.In both cases, the numbers of passenger single nucleotide variants per year were higher in PIGA-mutated clones than in PIGA-unmutated clones (16 vs. 11 mutations/year for Case 1 and 14 vs. 12 for Case 2). Assuming that variant acquisition rates per cell division are similar in both clones, the result suggests that PIGA-mutated clones had growth advantage against PIGA-unmutated clones.The acquisition of PIGA mutations was estimated to have occurred more than ten years prior to the PNH diagnosis. In Case 1, following the AA diagnosis, both PIGA-mutated clones and BCOR-mutated clones started expansion simultaneously, which suggests an association with immune attacks. Interestingly, even wild type clones expanded after AA diagnosis, suggesting bottle-neck effects of post-AA hematopoietic recovery. In Case 2, PIGA-mutated clones began expanding more than ten years before the AA diagnosis. Either a subclinical immune attack that might have existed before the diagnosis of clinical AA or the concomitant HMGA2 mutation conferred survival advantage for PIGA-mutated clones in Case 2. Conclusions: This estimation of the trajectories of the PIGA-mutated clone indicates that the pathogenesis of clonal expansion exhibits heterogeneity between two PNH patients.

Characterization of Hematopoietic Stem and Progenitor Cells of PNH Patients Using Single-Cell RNA Sequencing Analysis

The etiology of paroxysmal nocturnal hemoglobinuria (PNH) is somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs), and affected PNH-type cells lack cell surface expression of the glycosylphosphatidylinositol-anchored proteins (GPI-APs). In PNH, GPI-AP deficient cells clonally expand and occupy a large proportion of mature cells. Several hypotheses have been proposed to explain clonal expansion of PNH-type HSPCs, including reduced sensitivity to apoptosis and escape from immune-mediated cytotoxicity. However, definitive mechanisms remain unclear. To further understand PNH pathophysiology at high multi-dimensional resolution, we performed single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) cells from PNH patients. Fresh BM samples were obtained from eight PNH patients (male, n = 2, female, n = 6; median age, 51 [range, 21-67] years), who had large PNH-type granulocyte populations and stable clone sizes (median, 45 [range, 16-99] %), and five age- and sex-matched healthy donors (HDs). PNH-type and wild-type (WT) lineage-CD34+HSPCs were enriched from bone marrow mononuclear cells (BMMNCs) by flow cytometric sorting. Sorted cells were processed through the Chromium Controller (10x genomics, Pleasanton, CA, USA). scRNA-Seq libraries were constructed using the Chromium Single Cell 3' Reagent Kits (10x genomics) according to the manufacturer's instructions. The constructed libraries were sequenced with the NovaSeq 6000 system (Illumina, San Diego, CA, USA). We obtained 81,470 high-quality cells from PNH patients and 56,545 cells from HDs for further analysis. Cells were clustered with Seurat and visualized in UMAP. Cell clusters were assigned to 6 HSPC subsets based on expression of canonical marker genes (Figure 1A). To investigate compositional changes in BMMNCs between PNH patients and HDs, we performed differential abundance analysis with Milo and identified a prominent decrease in hematopoietic stem cells (HSCs) and lymphoid progenitors, as compared to HDs (Figure 1B). We then evaluated differential abundance between PNH-type and WT HSPCs in PNH patients, identifying no significant differences in each HSPC subsets (Figure 1B). These data were consistent with flow cytometric profiling of BMMNCs, similar proportions of PNH-type cells in each HSPC subsets in individual patients despite with profound decrease in PNH-type lymphocytes (Figure 1C). We next sought to investigate skewed gene sets in PNH-type HSPCs using gene set enrichment analysis. Comparison between PNH-type and WT HSPCs identified upregulation of inflammatory signaling pathways and downregulation of the cell-cycling pathways in PNH-type HSPCs, as compared to WT HSPCs (Figure 1D). We made comparisons within individual subsets. In HSCs, genes involved in IFN-γ response and oxidative phosphorylation were upregulated in PNH-type HSCs, while cell-cycling genes (ie. E2F targets and G2M checkpoint) were not dysregulated, as compared to WT HSCs. In summary,no significant skewing in lineage differentiation of PNH-type HSPCs was observed, suggesting PNH-type lymphocytes were negatively selected during the maturation process in lymphoid tissues. Upregulation of the inflammatory and immune response pathways in PNH-type HSPCs indicated a proinflammatory phenotype of PNH-type HSPCs. Upregulation of cell cycle-related pathways was not observed in PNH-type HSCs compared to WT HSCs, consistent with stable PNH clone sizes in our cohort of PNH patients.

Single-Cell Dissection Reveals a Distinct Origin of Small Paroxysmal Nocturnal Hemoglobinuria Clones in Immune Aplastic Anemia and Healthy Individuals

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) clones are characterized by the absence of cell surface glycosylphosphatidylinositol (GPI)-anchors owing to somatic mutations in the PIGA gene on the X chromosome. Large PNH clones derived from PIGA mutant multipotent progenitors (MPPs) result in clinically significant hemolysis. In immune aplastic anemia (AA), the PNH clone represents immune escape from T cell-mediated pathogenesis and is usually small (<1%), sometimes likely derived from lineage-committed progenitors (LCPs), with detectable GPI-anchor deficient (PNH-type) cell populations only in granulocytes and monocytes. Small PNH-type cells are also present in healthy individuals at a much lower frequency (<0.003%) than in AA patients. The nature of the small PNH-type cells is not fully understood because of the difficulties in analyzing rare cell populations. Methods In this study, we developed a method to detect PIGA mutations within a small population of cells at single-cell resolution. PNH-type cells were enriched from peripheral blood leukocytes using magnetic microbeads and monoclonal antibodies specific for GPI-anchored proteins. The enriched cell fraction was then subjected to fluorescence-activated cell sorting after staining with lineage-specific markers and fluorescein-labelled proaerolysin. We sorted a limited number (100 cells in males and 50 cells in females) of PNH-type granulocytes (PNH-Gs) and T cells (PNH-Ts) as well as wild-type cells into separate tubes, and their PIGA genes were directly amplified by multiplex PCR without a DNA extraction procedure for subsequent deep nucleotide sequencing. To ensure the specificity of PIGA mutation detection, we set cut-off thresholds of 2% for single-nucleotide substitutions and 1% for indels, in addition to the thresholds based on base-position-specific error rates in 10 sets of 2000 wild-type cells, as previously reported (Zaimoku et al. Blood 2021). Results In 19 sets of 50-100 wild-type cells from 13 individuals, we detected only 7 mutations with variant allele frequencies (VAFs) ranging from 2.1% to 4.2% (Figure 1). Three of these were synonymous mutations, and the remaining 4 were missense mutations, suggesting that these mutations may be true passenger mutations rather than sequencing errors. In all 6 healthy individuals, small PNH-type cells exhibited a polyclonal pattern. We detected numerous small, likely inactivating, PIGA mutations in PNH-Gs, with a median of 11 (range, 6-18) mutations per 100 cells. In 3 individuals assessed twice, approximately half of the PIGA mutations were detected in both sets of PNH-Gs, whereas the remaining mutations were unique to each set. Similarly, PNH-Ts from 4 healthy individuals showed polyclonality, with a median of 14 (range, 6-22) mutations per 100 cells. Interestingly, PIGA mutations were not shared between PNH-Gs and PNH-Ts, except for 2 splice site mutations with VAFs <10%. The polyclonal pattern was further confirmed by Sanger sequencing, which successfully detected 7 unique PIGA mutations in 5 sets of 5 PNH-type cells (instead of 100 cells) in a healthy donor, whereas no mutations were detected in 3 sets of 5 wild-type cells, except for 1 synonymous mutation. In contrast, small PNH-Gs of 5 AA patients, with frequencies of 0.02%, 0.08%, 0.09%, 0.2%, and 0.5% of total granulocytes, were primarily derived from single PIGA mutant clones, which accounted for 90%-99% of PNH-G populations. In another patient with AA, PNH-Gs with a frequency of 3.3% were derived from 2 dominant clones. Notably, the dominant PIGA mutations in the PNH-Gs of AA patients were all detectable in PNH-Ts, even in 2 patients whose PNH-Ts were not detectable (<0.003%) and in 3 patients whose percentages of PNH-Ts were 0.004%, 0.02%, and 0.08% before microbead-based enrichment. Discussion Our novel sequencing approach provides insight into the clonality and lineage distribution of small PNH-type cells without in vitro culture manipulation. In healthy individuals, most PNH clones originate from LCPs, where many cell divisions occur, thereby providing opportunities for the acquisition of PIGA mutations. In immune AA, only PIGA mutations acquired at the MPP level lead to clonal proliferation, strongly suggesting that the immune pathogenesis of AA targets MPPs, rather than LCPs. A clonality analysis of small PNH-type cells thus helps diagnose the immune pathogenesis of AA.

No Benefits from Glucocorticoid Maintenance Therapy in Patients with PNH

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, complement-mediated hemolytic anemia resulting from a somatic phosphatidylinositol glycan class A ( PIGA) mutation which bugs the biosynthesis of glycosylphosphatidylinositol (GPI). The lack of GPI-anchored proteins, especially CD55 and CD59 molecules, prompts blood cells to be prone to complement attack and lysis. Targeted on the terminal complement inhibitor has been recommended as a first-line treatment, such as eculizumab (C5 inhibitors), which not only controls hemolytic anemia, reduces thrombosis and blood transfusion, but also improves the staging status of chronic kidney disease in PNH patients. Complement inhibitors are not yet widely administrated by Chinese PNH patients, so glucocorticoids are still mostly used for the first-line treatment. However, the efficacy of glucocorticoid maintenance therapy in PNH is uncertain. Methods We conducted a prospective self-controlled comparison of 40 PNH patients with high disease activity (HDA), collecting anemia, hemolysis, thrombosis and renal function indicators at baseline and 1st, 3rd and 6th month after treatment in the Chinese Eastern Collaboration Group of Anemia (CECGA). All enrolled patients were administrated with prednisone 1.0 mg/kg/d at first and then adjusted the dosage according to the situation. The clinical trial registration number is ChiCTR2100050945. Results A total of 40 patients with PNH were enrolled in this study, 21 (52.5%, 21/40) were male, 19 (47.5%, 19/40) were female and the median age was 33.5 years (range 18-69 years). 29 of 40 (72.5%) presented classical PNH and 11 patients (27.5%) were diagnosed as PNH associated with bone marrow disease (BMD)-10 were aplastic anemia and 1 was myelodysplastic syndrome. Four cases detected single-site thrombosis and one had multi-site thrombosis. The most common locations were portal vein thrombus (2/6, 33.33%) and lower extremity intermuscular thrombus (2/6, 33.33%), others included mesentery vein, hepatic vein, splenic vein and intracranial microthrombus. The laboratory indexes at baseline were presented in Table 1. Compared to the baseline (Table 2), at 1 st, 3 rd, and 6 th month after treatment, both hemoglobin and red blood cell (RBC) have improved, but hemoglobin and RBC didn't keep rising, and showed no difference when comparing 3 rd month with 1 st month and 6 th month with 3 rd month. 19 patients (19/40, 47.5%) required blood transfusion before treatment, and 11 patients (11/40, 27.5%) needed transfusion at 1 st month ( P=0.021). Then 13 patients (13/40, 32.5%) at 3 rd month, 15 patients (15/40, 37.5%) at 6 th month were transfusion dependent ( P=0.109; P=0.388). After 6-month treatment, 3 cases of severe anemia (Hb < 30 g/L) at baseline were still severe anemia (3/7, 42.86%), and 4 cases (4/7, 57.14%) were upgraded to moderate anemia (60 g/L ≤ Hb ≤ 90 g/L). Among the original moderate anemia, 20 (20/31, 64.52%) cases were still moderate anemia, 6 (6/31, 19.35%) cases decreased to severe anemia and 5 (5/31, 16.13%) cases were improved to mild anemia (Hb > 90 g/L). 1 (1/2, 50%) patient of mild anemia originally maintained mid anemia and one (1/2, 50%) patient decreased to moderate anemia. The levels of LDH, indirect bilirubin (IBIL) and reticulocyte (Ret) were not changed significantly at 1 st, 3 rd, 6 th month after treatment. At 1 st, 3 rd, 6 th month, only 1, 2 and 2 patients achieved hemolytic control (LDH < 1.5 ULN). The d-dimer and glomerular filtration rate (GFR) also were indicated no difference. At 1 st, 3 rd, 6 th month, 3 (3/11, 27.27%), 4 (4/11, 36.36%), 6 (6/11, 54.54%) patients were back to normal range, 1 (1/11, 9.09%), 2 (2/11, 18.18%), 4 (4/11, 36.36%) patient was newly elevated (D-dimer > 0.55 mg/L) separately. At baseline 2 patients' renal function was severely reduced (GFR＜60ml/min/1.73m 2), and at 1 st, 3 rd, 6 th month, 1, 1, 2 patients had declined respectively. Discussions and Conclusions The use of prednisone in PNH patients with HDA may initially improve anemia, but the improvement in anemia may also be due to self-remission after paroxysmal episodes of PNH itself, or transfusion. The maintenance therapy with prednisone cannot control hemolysis, improve thrombosis and renal function damage. Long-term glucocorticoid maintenance therapy for PNH is not recommended.

155 T cell deficiency associated with downregulation of GPI-anchored proteins

155 T cell deficiency associated with downregulation of GPI-anchored proteins

Promiscuous Artemisia annua glycosylphosphatidylinositol-anchored lipid transfer protein 1 (AaLTPG1) promotes terpenoids accumulation in planta

LTPG, encoded by a glycosylphosphatidylinositol-anchored lipid transfer protein family gene, plays a role in exporting fatty acids to the plant surface through the hydrophilic cell wall layer. While soluble extracellular LTPs are known to function in terpenoid accumulation in trichomes, whether membrane-integrated LTPG influences terpenoid accumulation and its underlying mechanisms remains poorly understood. In this study, we report an Artemisia annua LTPG1, which is strongly expressed in floral tissues and positively correlated with three artemisinin biosynthetic genes. The recombinant AaLTPG1 displayed promiscuous binding activity with fatty acids and artemisinin precursors in vitro, resulting in an increase in terpenoids and lipids contents in both AaLTPG1 transgenic tobacco without altering trichome initiation, development, or secretion. Transcriptome sequencing further revealed that ethylene responsive genes were repressed in AaLTPG1-overexpressing tobacco plants. Consistently, artemisinin and its precursors were found to increase transiently in A. annua leaves, and the expression level of ethylene-responsive factors (ERFs) was down-regulated in A. annua. Collectively, this work illustrates the positive regulatory role of LTPG in terpenoids and lipids accumulation by exporting hydrophobic metabolites and repressing ethylene signaling, making AaLTPG1 a potential ideal target for metabolic engineering to improve artemisinin accumulation.

The Mechanism of Transcription Factor Swi6 in Regulating Growth and Pathogenicity of Ceratocystis fimbriata: Insights from Non-Targeted Metabolomics.

Ceratocystis fimbriata (C. fimbriata) is a notorious pathogenic fungus that causes sweet potato black rot disease. The APSES transcription factor Swi6 in fungi is located downstream of the cell wall integrity (CWI)-mitogen-activated protein kinase (MAPK) signaling pathway and has been identified to be involved in cell wall integrity and virulence in several filamentous pathogenic fungi. However, the specific mechanisms by which Swi6 regulates the growth and pathogenicity of plant pathogenic fungi remain elusive. In this study, the SWI6 deletion mutants and complemented strains of C. fimbriata were generated. Deletion of Swi6 in C. fimbriata resulted in aberrant growth patterns. Pathogenicity assays on sweet potato storage roots revealed a significant decrease in virulence in the mutant. Non-targeted metabolomic analysis using LC-MS identified a total of 692 potential differentially accumulated metabolites (PDAMs) in the ∆Cfswi6 mutant compared to the wild type, and the results of KEGG enrichment analysis demonstrated significant enrichment of PDAMs within various metabolic pathways, including amino acid metabolism, lipid metabolism, nucleotide metabolism, GPI-anchored protein synthesis, and ABC transporter metabolism. These metabolic pathways were believed to play a crucial role in mediating the growth and pathogenicity of C. fimbriata through the regulation of CWI. Firstly, the deletion of the SWI6 gene led to abnormal amino acid and lipid metabolism, potentially exacerbating energy storage imbalance. Secondly, significant enrichment of metabolites related to GPI-anchored protein biosynthesis implied compromised cell wall integrity. Lastly, disruption of ABC transport protein metabolism may hinder intracellular transmembrane transport. Importantly, this study represents the first investigation into the potential regulatory mechanisms of SWI6 in plant filamentous pathogenic fungi from a metabolic perspective. The findings provide novel insights into the role of SWI6 in the growth and virulence of C. fimbriata, highlighting its potential as a target for controlling this pathogen.

The Arabidopsis GPI-anchored protein COBL11 is necessary for regulating pollen tube integrity

Pollen tube integrity is required for achieving double fertilization in angiosperms. The rapid alkalinization factor4/19-ANXUR1/2-Buddha's paper seal 1/2 (RALF4/19-ANX1/2-BUPS1/2)-complex-mediated signaling pathway is critical to maintain pollen tube integrity, but the underlying mechanisms regulating the polar localization and distribution of these complex members at the pollen tube tip remain unclear. Here, we find that COBRA-like protein 11 (COBL11) loss-of-function mutants display a low pollen germination ratio, premature pollen tube burst, and seed abortion in Arabidopsis. COBL11 could interact with RALF4/19, ANX1/2, and BUPS1/2, and COBL11 functional deficiency could result in the disrupted distribution of RALF4 and ANX1, altered cell wall composition, and decreased levels of reactive oxygen species in pollen tubes. In conclusion, COBL11 is a regulator of pollen tube integrity during polar growth, which is conducted by a direct interaction that ensures the correct localization and polar distribution of RALF4 and ANX1 at the pollen tube tip.

In Silico Evaluation, Phylogenetic Analysis, and Structural Modeling of the Class II Hydrophobin Family from Different Fungal Phytopathogens.

The class II hydrophobin group (HFBII) is an extracellular group of proteins that contain the HFBII domain and eight conserved cysteine residues. These proteins are exclusively secreted by fungi and have multiple functions with a probable role as effectors. In the present study, a total of 45 amino acid sequences of hydrophobin class II proteins from different phytopathogenic fungi were retrieved from the NCBI database. We used the integration of well-designed bioinformatic tools to characterize and predict their physicochemical parameters, novel motifs, 3D structures, multiple sequence alignment (MSA), evolution, and functions as effector proteins through molecular docking. The results revealed new features for these protein members. The ProtParam tool detected the hydrophobicity properties of all proteins except for one hydrophilic protein (KAI3335996.1). Out of 45 proteins, six of them were detected as GPI-anchored proteins by the PredGPI server. Different 3D structure templates with high pTM scores were designed by Multifold v1, AlphaFold2, and trRosetta. Most of the studied proteins were anticipated as apoplastic effectors and matched with the ghyd5 gene of Fusarium graminearum as virulence factors. A protein-protein interaction (PPI) analysis unraveled the molecular function of this group as GTP-binding proteins, while a molecular docking analysis detected a chitin-binding effector role. From the MSA analysis, it was observed that the HFBII sequences shared conserved 2 Pro (P) and 2 Gly (G) amino acids besides the known eight conserved cysteine residues. The evolutionary analysis and phylogenetic tree provided evidence of episodic diversifying selection at the branch level using the aBSREL tool. A detailed in silico analysis of this family and the present findings will provide a better understanding of the HFBII characters and evolutionary relationships, which could be very useful in future studies.

The lipoprotein lipase that is shuttled into capillaries by GPIHBP1 enters the glycocalyx where it mediates lipoprotein processing.

Lipoprotein lipase (LPL), the enzyme that carries out the lipolytic processing of triglyceride-rich lipoproteins (TRLs), is synthesized by adipocytes and myocytes and secreted into the interstitial spaces. The LPL is then bound by GPIHBP1, a GPI-anchored protein of endothelial cells (ECs), and transported across ECs to the capillary lumen. The assumption has been that the LPL that is moved into capillaries remains attached to GPIHBP1 and that GPIHBP1 serves as a platform for TRL processing. In the current studies, we examined the validity of that assumption. We found that an LPL-specific monoclonal antibody (mAb), 88B8, which lacks the ability to detect GPIHBP1-bound LPL, binds avidly to LPL within capillaries. We further demonstrated, by confocal microscopy, immunogold electron microscopy, and nanoscale secondary ion mass spectrometry analyses, that the LPL detected by mAb 88B8 is located within the EC glycocalyx, distant from the GPIHBP1 on the EC plasma membrane. The LPL within the glycocalyx mediates the margination of TRLs along capillaries and is active in TRL processing, resulting in the delivery of lipoprotein-derived lipids to immediately adjacent parenchymal cells. Thus, the LPL that GPIHBP1 transports into capillaries can detach and move into the EC glycocalyx, where it functions in the intravascular processing of TRLs.

NAC transcription factor SlNOR-like1 plays a dual regulatory role in tomato fruit cuticle formation.

The plant cuticle is an important protective barrier on the plant surface, constructed mainly by polymerized cutin matrix and a complex wax mixture. Although the pathway of plant cuticle biosynthesis has been clarified, knowledge of the transcriptional regulation network underlying fruit cuticle formation remains limited. In the present work, we discovered that tomato fruits of the NAC transcription factor SlNOR-like1 knockout mutants (nor-like1) produced by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] displayed reduced cutin deposition and cuticle thickness, with a microcracking phenotype, while wax accumulation was promoted. Further research revealed that SlNOR-like1 promotes cutin deposition by binding to the promoters of glycerol-3-phosphate acyltransferase6 (SlGPAT6; a key gene for cutin monomer formation) and CUTIN DEFICIENT2 (SlCD2; a positive regulator of cutin production) to activate their expression. Meanwhile, SlNOR-like1 inhibits wax accumulation, acting as a transcriptional repressor by targeting wax biosynthesis, and transport-related genes 3-ketoacyl-CoA synthase1 (SlKCS1), ECERIFERUM 1-2 (SlCER1-2), SlWAX2, and glycosylphosphatidylinositol-anchored lipid transfer protein 1-like (SlLTPG1-like). In conclusion, SlNOR-like1 executes a dual regulatory effect on tomato fruit cuticle development. Our results provide a new model for the transcriptional regulation of fruit cuticle formation.

Structural Analysis of GPI-glycans from GPI-anchored Proteins by Mass Spectrometry

Structural Analysis of GPI-glycans from GPI-anchored Proteins by Mass Spectrometry

Structural Analysis of GPI-glycans from GPI-anchored Proteins by Mass Spectrometry

Structural Analysis of GPI-glycans from GPI-anchored Proteins by Mass Spectrometry

Engineered CD4 T cells expressing a membrane anchored viral inhibitor restrict HIV-1 through cis and trans mechanisms.

HIV-1 infection of target cells can occur through either cell-free virions or cell-cell transmission in a virological synapse, with the latter mechanism of infection reported to be 100- to 1,000-fold more efficient. Neutralizing antibodies and entry inhibitors effectively block cell-free HIV-1, but with few exceptions, they display much less inhibitory activity against cell-mediated HIV-1 transmission. Previously, we showed that engineering HIV-1 target cells by genetically linking single-chain variable fragments (scFvs) of antibodies to glycosyl phosphatidylinositol (GPI) potently blocks infection by cell-free virions and cell-mediated infection by immature dendritic cell (iDC)-captured HIV-1. Expression of scFvs on CD4+ cell lines by transduction with X5 derived anti-HIV-1 Env antibody linked to a GPI attachment signal directs GPI-anchored scFvs into lipid rafts of the plasma membrane. In this study, we further characterize the effect of GPI-scFv X5 on cell-cell HIV-1 transmission from DCs to target cells. We report that expression of GPI-scFv X5 in transduced CD4+ cell lines and human primary CD4+ T cells potently restricts viral replication in iDC- or mDC-captured HIV-1 in trans. Using live-cell imaging, we observed that when GPI-GFP or GPI-scFv X5 transduced T cells are co-cultured with iDCs, GPI-anchored proteins enrich in contact zones and subsequently migrate from T cells into DCs, suggesting that transferred GPI-scFv X5 interferes with HIV-1 infection of iDCs. We conclude that GPI-scFv X5 on the surface of transduced CD4+ T cells not only potently blocks cell-mediated infection by DCs, but it transfers from transduced cells to the surface of iDCs and neutralizes HIV-1 replication in iDCs. Our findings have important implications for HIV-1 antibody-based immunotherapies as they demonstrate a viral inhibitory effect that extends beyond the transduced CD4+ T cells to iDCs which can enhance HIV-1 replication.

Competition for calnexin binding regulates secretion and turnover of misfolded GPI-anchored proteins.

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.

Negr1-Derived Peptides Trigger ALK Degradation and Halt Neuroblastoma Progression In Vitro and In Vivo.

Neuroblastoma is among the most common childhood cancers. Neuroblastoma in advanced stages is one of the most intractable pediatric cancers, notwithstanding the recent therapeutic advances. ALK mutations are among the leading cause of hereditary neuroblastoma and account for more than 14% of the somatically acquired alterations. ALK kinase activity is currently one of the main targets for pharmacological strategies. However, evidence from ALK fusion-positive lung cancer studies has shown that resistance to ALK inhibition arises during the therapy, causing a relapse within several years. IgLONs are membrane-bound proteins involved in cell-to-cell adhesion. The expression of the IgLON family results altered in different cancers. We found that the IgLON member Negr1 is downregulated in neuroblastoma. The ectopic overexpression of Negr1 impairs neuroblastoma growth in vitro and in vivo. Negr1 exists as a GPI-anchored membrane-bound protein and as a soluble protein released upon metalloprotease cleavage. We generated and characterized a panel of Negr1-derived peptides. The treatment with Negr1 protein and derived peptides induce ALK downregulation and halt neuroblastoma progression in vitro and in vivo.

Structures of liganded glycosylphosphatidylinositol transamidase illuminate GPI-AP biogenesis

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.

Paroxysmal Nocturnal Hemoglobinuria: Biology and Treatment.

Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal hematopoietic disorder characterized by the lack of glycosylphosphatidylinositol-anchored proteins (GPI-APs) as a consequence of somatic mutations in the phosphatidylinositol glycan anchor biosynthesis class A (PIGA) gene. Clinical manifestations of PNH are intravascular hemolysis, thrombophilia, and bone marrow failure. Treatment of PNH mainly relies on the use of complement-targeted therapy (C5 inhibitors), with the newest agents being explored against other factors involved in the complement cascade to alleviate unresolved intravascular hemolysis and extravascular hemolysis. This review summarizes the biology and current treatment strategies for PNH with the aim of reaching a general audience with an interest in hematologic disorders.

Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis.

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.

Comprehensive in silico analysis of glycosylphosphatidylinositol- anchored protein (GPI-AP) related genes expression profiles in human normal and cancer tissues.

In human cells, there are more than 146 glycosylphosphatidylinositol-anchored proteins (GPI-APs), including receptors, ligands, adhesion molecules and enzymes. The proteins are associated with membrane microdomains called lipid rafts through GPI, and plays a variety of important biological functions. At present, plenty of studies have been carried out on the biosynthesis of GPI-APs. The biosynthesis of GPI-APs requires at least 20 steps, and more than 40 GPI biosynthetic genes have been identified. However, it remains unclear how expression of GPI-AP related genes is regulated in normal and cancer tissues. In this study, we utilized gene expression data from both the TCGA database and GTEx portal to analysis the gene expression involved in GPI-AP biosynthesis and encoding GPI-APs in normal and cancer tissues. In order to perform a comprehensive analysis, we employed the GlycoMaple, a tool that is specifically designed to analyze glycosylation pathways. The results showed that compared with normal tissues, the expression of genes involved in GPI-AP biosynthesis in cancer tissues such as early glioma, glioblastoma multiforme, pancreatic cancer, testicular germ cell carcinoma, skin primary cutaneous melanoma and skin metastatic cutaneous melanoma, was changed significantly. Particularly, the expression of PIGY in these six cancers was increased. In addition, the expression of CD14, a GPI-AP gene, was increased in these six cancers. The expression of GAS1, GPC2 and GPC4 was increased only in early glioma and glioblastoma multiforme indicating that some GPI-APs such as GAS1 can be used as biomarkers of glioma. This study provides new insights into the expression of GPI-AP related genes in normal and cancer tissues, and lays a solid foundation for the development of GPI-APs as biomarkers.