Anthrax Toxin Receptor Research Articles

A candidate transacting modulator of fetal hemoglobin gene expression in the Arab-Indian haplotype of sickle cell anemia.

Fetal hemoglobin (HbF) levels are higher in the Arab-Indian (AI) β-globin gene haplotype of sickle cell anemia compared with African-origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes-seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34+ cells. As CD34+ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118-1122, 2016. © 2016 Wiley Periodicals, Inc.

Fell-Muir Lecture: Regulatory mechanisms of skeletal and connective tissue development and homeostasis - lessons from studies of human disorders.

Studies of proliferative hemangiomas have led to the discovery that interactions of endothelial cells with extracellular matrix and/or Vascular Endothelial Growth Factor (VEGF)-A stimulate the expression of VEGFR1, the VEGF decoy receptor, and suppress VEGF-dependent VEGFR2 signalling by a mechanism that requires the matrix-binding receptor Anthrax Toxin Receptor (ANTXR)1, VEGFR2, β1 integrin and the Nuclear Factor of Activated T cells (NFAT). In hemangioma endothelial cells, all these components are present, but are functionally compromised, so that the levels of VEGFR1 are extremely low and VEGFR2 signalling is constitutively active. Consequently, the levels of Hypoxia Inducible Factor (HIF)-1α and its transcriptional targets, VEGF-A and C-X-C motif chemokine 12 (CxCl12), are elevated and a positive VEGF-A feedback loop is established. Overexpression of ANTXR1, carrying a heterozygous Ala-to-Thr mutation, induces hemangioma-like signalling in control endothelial cells; VEGF signalling is normalized when wild-type ANTXR1 is overexpressed in hemangioma cells. These findings suggest that ANTXR1 functions as a negative regulator of VEGF-A signalling. Studies of a mouse model of the Growth Retardation, Alopecia, Pseudo-anodontia and Optic Atrophy (GAPO) syndrome, caused by the loss-of-function mutations in ANTXR1, as well as knock-in mice carrying the Ala-to-Thr ANTXR1 mutation, confirm that ANTXR1 functions as a suppressor of VEGF-A signalling. Cutaneous endothelial cells isolated from ANTXR1-deficient mice exhibit low levels of VEGFR1, elevated levels of VEGF-A, HIF-1α and CxCl12 and activated VEGFR2 signalling as in hemangioma. Increased numbers of myeloid cells in the skin of ANTXR1-deficient mice are associated with reduced vascularity and increased skin fibrosis, suggesting a mechanism for hemangioma involution and replacement by fibrotic scars. Through controlling VEGF-A signalling and extracellular matrix synthesis, ANTXR1 is emerging as a key regulator of skeletal and connective tissue development and homeostasis.

Abstract 4356: Identification of the anthrax toxin receptor (ANTXR1) as the high affinity cellular receptor for Seneca Valley Virus (SVV)

Abstract Small cell lung cancer (SCLC) is an extremely aggressive and almost universally lethal form of lung cancer, which accounts for approximately 15% of all lung cancer diagnoses annually. Novel and more effective therapies are desperately needed for SCLC, as the standard treatment has not improved in almost three decades. Recently, oncolytic viruses have shown strong anti-cancer efficacies in tumors with high levels of selectivity for cancer cells. The oncolytic picornavirus, Seneca Valley Virus (SVV), has previously been shown to infect and lyse cancer cells with neuroendocrine features, notably SCLC and pediatric brain tumors. Pre-clinical mouse models and early phase clinical trials have confirmed the ability of SVV to home directly to the tumor through the vasculature, specifically infect tumor cells without affecting normal cells, and replicate to high viral titers intratumorally even after the production of neutralizing antibodies. However, the clinical development of SVV has been hindered by the inability to identify patients who would benefit from SVV virotherapy because essential host proteins for viral entry, including the cellular receptor, have not been characterized. Using genome wide loss-of-function CRISPR screens in the SCLC cell line H446 and haploid cell line HAP1, we identified the anthrax toxin receptor 1 (ANTXR1) as an essential host protein for SVV. Interestingly, ANTXR1 is also one of the receptors for the toxin of Bacillus anthracis and has been previously described as being upregulated in tumor endothelial cells. Secondary screens performed in both cell lines confirmed gene knockout of ANTXR1 conferred resistance to SVV. We established that in multiple permissive SCLC and pediatric cancer cell lines that loss of ANTXR1 protein expression using CRISPR gene knockout leads to a loss of SVV permissivity. Re-expression of ANTXR1 in ANTXR1 knockout cell lines was sufficient to rescue SVV permissivity. Furthermore, the expression of the ANTXR1 protein in two non-permissive SCLC cell lines led to a conversion in SVV permissivity. A direct and high affinity interaction was confirmed between SVV and ANTXR1 using co-immunoprecipitation assays. Additionally, we discovered that ANTXR1 is the major binding determinant for SVV on cells using a binding assay with fluorescently labeled SVV. Lastly we determined using previously published gene expression data from the Cancer Cell Line Encyclopedia (CCLE) that non-permissive lines which do express ANTXR1 can be explained by the presence of an intact and active innate immune response pathway. These findings identify ANTXR1 as the high-affinity cellular receptor for SVV. Furthermore, these data identify a predictive biomarker for SVV permissivity that could lead to defined selection criteria for subsequent clinical trials testing SVV in both SCLC and pediatric brain cancer patients. Citation Format: Linde A. Miles, J.T. Poirier, Charles M. Rudin. Identification of the anthrax toxin receptor (ANTXR1) as the high affinity cellular receptor for Seneca Valley Virus (SVV). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4356.

Pharmacology and Anti-infective Role of Raxibacumab: A Novel Monoclonal Antibody for the Treatment of Anthrax.

Anthrax, caused by Bacillus anthracis (B anthracis), poses a potential threat as a bioterror agent because after inhalation, the spores rapidly cause bacteraemia and toxaemia. It produces a toxin consisting of three proteins ie protective antigen (PA), oedema factor (EF) and lethal factor (LF). Protective antigen plays a central role in the pathophysiology of anthrax and offers an excellent therapeutic target for treatment of anthrax. Raxibacumab is a recombinant, fully human, IgG1λ monoclonal antibody directed against PA of B anthracis. It inhibits PA binding to the anthrax toxin receptor and inhibits toxin-mediated cell death. It has been approved under animal rule or animal efficacy rule by the United States Food and Drug Administration which comes into play when it is not feasible or ethical to perform controlled clinical trials in humans. It has shown promising results in various animal studies which includes significantly improved survival rates in the raxibacumab group than non-raxibacumab group.

The Ins and Outs of Anthrax Toxin.

Anthrax is a severe, although rather rare, infectious disease that is caused by the Gram-positive, spore-forming bacterium Bacillus anthracis. The infectious form is the spore and the major virulence factors of the bacterium are its poly-γ-D-glutamic acid capsule and the tripartite anthrax toxin. The discovery of the anthrax toxin receptors in the early 2000s has allowed in-depth studies on the mechanisms of anthrax toxin cellular entry and translocation from the endocytic compartment to the cytoplasm. The toxin generally hijacks the endocytic pathway of CMG2 and TEM8, the two anthrax toxin receptors, in order to reach the endosomes. From there, the pore-forming subunit of the toxin inserts into endosomal membranes and enables translocation of the two catalytic subunits. Insertion of the pore-forming unit preferentially occurs in intraluminal vesicles rather than the limiting membrane of the endosome, leading to the translocation of the enzymatic subunits in the lumen of these vesicles. This has important consequences that will be discussed. Ultimately, the toxins reach the cytosol where they act on their respective targets. Target modification has severe consequences on cell behavior, in particular on cells of the immune system, allowing the spread of the bacterium, in severe cases leading to host death. Here we will review the literature on anthrax disease with a focus on the structure of the toxin, how it enters cells and its immunological effects.

Roles of Anthrax Toxin Receptor 2 in Anthrax Toxin Membrane Insertion and Pore Formation.

Interaction between bacterial toxins and cellular surface receptors is an important component of the host-pathogen interaction. Anthrax toxin protective antigen (PA) binds to the cell surface receptor, enters the cell through receptor-mediated endocytosis, and forms a pore on the endosomal membrane that translocates toxin enzymes into the cytosol of the host cell. As the major receptor for anthrax toxin in vivo, anthrax toxin receptor 2 (ANTXR2) plays an essential role in anthrax toxin action by providing the toxin with a high-affinity binding anchor on the cell membrane and a path of entry into the host cell. ANTXR2 also acts as a molecular clamp by shifting the pH threshold of PA pore formation to a more acidic pH range, which prevents premature pore formation at neutral pH before the toxin reaches the designated intracellular location. Most recent studies have suggested that the disulfide bond in the immunoglobulin (Ig)-like domain of ANTXR2 plays an essential role in anthrax toxin action. Here we will review the roles of ANTXR2 in anthrax toxin action, with an emphasis on newly updated knowledge.

Anthrax Toxin Receptor 1 Is Essential for Arteriogenesis in a Mouse Model of Hindlimb Ischemia.

Anthrax toxin receptor 1/tumor endothelial marker 8 (Antxr1 or TEM8) is up-regulated in tumor vasculature and serves as a receptor for anthrax toxin, but its physiologic function is unclear. The objective of this study was to evaluate the role of Antxr1 in arteriogenesis. The role of Antxr1 in arteriogenesis was tested by measuring gene expression and immunohistochemistry in a mouse model of hindlimb ischemia using wild-type and ANTXR1-/- mice. Additional tests were performed by measuring gene expression in in vitro models of fluid shear stress and hypoxia, as well as in human muscle tissues obtained from patients having peripheral artery disease. We observed that Antxr1 expression transiently increased in ischemic tissues following femoral artery ligation and that its expression was necessary for arteriogenesis. In the absence of Antxr1, the mean arterial lumen area in ischemic tissues decreased. Antxr1 mRNA and protein expression was positively regulated by fluid shear stress, but not by hypoxia. Furthermore, Antxr1 expression was elevated in human peripheral artery disease requiring lower extremity bypass surgery. These findings demonstrate an essential physiologic role for Antxr1 in arteriogenesis and peripheral artery disease, with important implications for managing ischemia and other arteriogenesis-dependent vascular diseases.

A complex role of anthrax toxin receptor 2 polymorphisms and capillary morphogenesis protein 2 in ankylosing spondylitis pathogenesis.

This study investigated the role of anthrax toxin receptor 2 (ANTXR2) gene polymorphisms and capillary morphogenesis protein 2 (CMG2) expression in susceptibility and pathogenesis to ankylosing spondylitis (AS) in the Han Chinese in Beijing. A case-control study was performed using 602 AS patient samples meeting the revised New York criterion and 619 matched controls from Han Chinese individuals. Nineteen single-nucleotide polymorphisms (SNPs) of ANTXR2 genes were selected and genotyped using the Sequenom iPlex platform. Real-time polymerase chain reaction and flow cytometry were performed to investigate the impact of SNP polymorphisms on ANTXR2 transcription and CMG2 expression, respectively. The association of variants with AS was examined with UNPHASED 3.1.5. A novel association was observed between AS and three SNPs in the ANTXR2 gene (rs4690127, rs6823031, and rs4333130; P = 0.004, 0.011, and 0.013, respectively), confirming the association between rs433130 and AS in the Han Chinese. The strongest haplotype association was observed with rs4690127-rs6823031-rs4333130 (P = 2.5 × 10(-4)). rs6534639 and rs4333130 showed a cis-interaction (P = 0.027) in AS. ANTXR2 messenger RNA (mRNA) expression was significantly higher in the AS group than in the control group (P = 0.039). CMG2 expression in the lipopolysaccharide (LPS)-stimulated group was significantly lower than that in the control group (P = 0.018). This study reports a novel association between ANTXR2 and AS in the Han Chinese. ANTXR2 genetic polymorphisms affect ANTXR2 mRNA transcription and CMG2 expression. The opposing results observed for ANTXR2 transcription and CMG2 expression suggest a complex role of ANTXR2 polymorphisms in AS pathogenesis.

ANTXR-1 and -2 independent modulation of a cytotoxicity mediated by anthrax toxin in human cells.

Several animal models have shown that anthrax toxin (ATX) elicits a cytotoxic effect on host cells through anthrax toxin receptor (ANTXR) function. In this study, compared with mouse cells, cells obtained from humans exhibited low sensitivity to ATX-mediated cytotoxicity, and the sensitivity was not correlated with expression levels of ANTXRs. ATX treatment also induced a cytotoxic effect in other cultured human cells, human embryonic kidney (HEK) 293 cells, that express ANTXRs at undetectable levels. Furthermore, ectopic expression of ANTXRs in HEK293 cells did not affect the sensitivity to ATX treatment. These findings suggest that there is an ANTXR-independent cytotoxic mechanism in human cells.

Anthrax Susceptibility: Human Genetic Polymorphisms Modulating ANTXR2 Expression.

Anthrax toxin causes anthrax pathogenesis and expression levels of ANTXR2 (anthrax toxin receptor 2) are strongly correlated with anthrax toxin susceptibility. Previous studies found that ANTXR2 transcript abundance varies considerably in individuals of different ethnic/geographical groups, but no eQTLs (expression quantitative trait loci) have been identified. By using 3C (chromatin conformation capture), CRISPR-mediated genomic deletion and dual-luciferase reporter assay, gene loci containing cis-regulatory elements of ANTXR2 were localized. Two SNPs (single nucleotide polymorphism) at the conserved CREB-binding motif, rs13140055 and rs80314910 in the promoter region of the gene, modulating ANTXR2 promoter activity were identified. Combining these two regulatory SNPs with a previously reported SNP, rs12647691, for the first time, a statistically significant correlation between human genetic variations and anthrax toxin sensitivity was observed. These findings further our understanding of human variability in ANTXR2 expression and anthrax toxin susceptibility.

Molecular assembly of lethal factor enzyme and pre-pore heptameric protective antigen in early stage of translocation.

During intoxication, the anthrax toxin lethal (LF) and edema (EF) factors initially assemble with the protective antigen (PA) on the plasma membrane of cells expressing the membrane-bound surface-exposed anthrax toxin receptor (ATR). This takes place at the physiological pH prior to entering the acidic environment of the endosome. We elucidated the molecular dynamics (MD) behaviors of the three-dimensional structure of the (PA63)7LF3 complex in various conformations and analyzed the dynamical properties of the fully loaded pre-pore complex on the plasma membrane at the physiological pH. The analysis points to the interaction networks of amino acids conserved between PA63 octamer and heptamer, which are not affected during the initial stage of the LFs binding. The simulations show an asymmetrical movement of the complex domains that directly affect LFs conformations. The conformational and structural alterations of the 2β2-2β3 loops of PA subunits are associated with pore formation. The early conformational changes of the loops appear as they peel off from the domain 2 toward domain 4 of each PA subunit. The LFs unfold in 1α1 segments of their N-terminal initiating the early stage of the pre-pore formation. The results indicate instable regions within the complex and provide important clues concerning the detail of fluctuating residues of the LF-PA interface regions at the early steps of toxins translocation.

A Candidate Trans-Acting Modulator of Fetal Hemoglobin Gene Expression in the Arab-Indian Haplotype of Sickle Cell Anemia

In the Arab-Indian (AI) β-globin gene (HBB) haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are higher and the disease phenotype milder than in African HBB haplotypes. To study the genetic basis of HbF regulation in the AI haplotype, whole genome sequencing (WGS) was done in 14 unrelated Saudi AI haplotype HbS homozygotes; 7 selected for low HbF (7.9±1.9%) and 7 for high HbF (18.5±3.2%). WGS was also done in 3 Indians with the AI haplotype (HbF 26.0±4.5%), 3 African Americans with the Benin haplotype selected because of the uncommonly high HbF for this haplotype (19.8±0.4%) and 1 African American homozygote with the Senegal haplotype (HbF 16.0%). Association of SNPs with HbF was tested using simple linear regression with an additive genetic model, and the Sequence Kernel Association Test using sliding windows of 5 SNPs. We found 465 SNPs that were significantly associated with HbF in a single SNP analysis (p≤10-5). Following annotation using SNPper and ENCODE RegulomeDB, PLINK was employed to remove all SNPs in high linkage disequilibrium (r2>0.8) ending with 128 SNPs for follow-up analysis. These prioritized variants were further investigated using functional evaluation tools such as SCAN, Hembase, ErythronDB and the results of WGS were followed by genome-wide SNP analysis, imputation of genotypes to 1000genomes, and targeted direct genotyping in the following cohorts with sickle cell anemia: 110 AI haplotype cases (HbF 18.0±7.0%); 71 Saudi Benin haplotype cases (HbF 11.4±4.1%); 44 Indian AI haplotype homozygotes (HbF 23.0±4.8%); 894 African Americans with diverse HBB haplotypes (HbF 5.2±5.6%). All cases were examined when the HbF levels had stabilized. None of the patients were using hydroxyurea when HbF was measured. Based on WGS, 2 SNPs, rs4527238 and rs35685045 (D'=1) in intron 9 of ANTXR1 (anthrax toxin receptor 1, 2p13.1) remained associated with HbF after correction for multiple comparisons. This result was replicated in the cohort of 110 additional AI haplotype Saudi patients but not in cohorts with other HBB haplotypes. The alternative alleles of these 2 SNPs had nearly equal frequencies in Saudis with the Saudi AI haplotype and lower frequencies in Indians, Saudi Benin haplotype and African Americans with sickle cell anemia. The T allele of rs4527238 and the C allele of rs35685045 are associated with high HbF. ANTXR1 SNPs explained 10% of the HbF variability compared with 8% for BCL11A; these genes had independent, additive effects on HbF and explained about 15% of HbF variability. RNA sequencing and gene expression microarray analysis at 3 time points during the erythroid differentiation of CD34+ and iPSCs isolated from the same subjects showed that only the long splice variant of ANTXR1 was expressed. Expression increased over time in CD34+ erythroid cells and decreased over time in iPSC-derived erythroid progenitors in a pattern similar to BCL11A expression, with increasing expression as CD34+ cells matured and HbF expression fell and declining expression as iPSCs differentiated and HbF expression increased. Immunofluorescence co-staining for ANTXR1 and HbF during the erythroid differentiation of sickle iPSCs showed that reduced expression of ANTXR1 was paralleled by increased expression of HbF. By GTEx analysis, eQTLs at rs4527238 and rs35685045 showed a trend for increased expression according to genotype with rs4527238 T/T having the lowest level of expression and T/C and C/C genotypes, higher expression levels (p= 0.08) and for rs35685045, C/C the lowest expression levels and C/T and T/T higher expression levels (p= 0.07); rs4527238 and rs35685045 had no effect on the expression of BCL11A, a repressor of HbF expression located at 2p16 (p=0.8). STRING 9.1 predicted protein interactions of ANTXR1 directly with LRP6, HDAC2, BCLX and BRAC1 with the highest level of confidence (0.95) with LRP6. The HbF phenotype in AI haplotype sickle cell anemia is unique and ANTXR1 is the first trans-acting element associated with HbF whose effects appear to be limited to the Saudi AI haplotype. ANTXR1 might have dual effects on HbF, indirectly affecting hematopoiesis via interactions with LRP6 and Wnt signaling, and directly modulating HBG expression through its effects on HDAC2. A dual effect on HbF expression has been suggested for MYB. Whether targeting ANTXR1 is a therapeutic option will require further studies. DisclosuresNo relevant conflicts of interest to declare.

Determination of IL1 R2, ANTXR2, CARD9, and SNAPC4 single nucleotide polymorphisms in Iranian patients with ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory disease of unknown origin, while both genetic and environmental factors have been demonstrated to be etiologically involved. Recent genome-wide association and replication studies have suggested that anthrax toxin receptor 2 (ANTXR2), interleukin-1 receptor 2 (IL1R2), caspase recruitment domain-containing protein 9 (CARD9), and small nuclear RNA-activating complex polypeptide 4 (SNAPC4) seem to be associated with AS pathogenesis. This case-control study was performed on 349 unrelated AS patients and 469 age- and gender-matched healthy controls, to investigate whether these non-MHC genes (IL1R2 rs2310173, ANTXR2 rs4333130, CARD9 rs4077515, and SNAPC4 rs3812571) influence the AS risk in Iranian population. ANTXR2 rs4333130 allele C (p = 0.0328; OR 0.744, 95% CI 0.598-0.927) and genotype CC (p = 0.0108; OR 0.273, 95% CI 0.123-0.605) were found to be significantly protective against AS. No other associations were found between AS and studied genes. The association between ANTXR2 rs4333130 and AS was independent of HLA-B27 status. Moreover, we found clinical disease severity scores (BASDAI and BASFI) and pain score were higher in ANTXR2 rs4333130 CT genotype. However, we observed that CARD9 allele C (p = 0.012) and genotype CC (p = 0.012) were significant protective factors against AS only in HLA-B27-negative patients, and IL1R2 rs2310173 genotype GT was mildly protective against AS only in HLA-B27-negative status. These findings support the role of non-MHC pathogenic pathways in susceptibility to AS and warrants more comprehensive studies focusing on these non-MHC pathways for developing novel therapeutic strategies.

Insights Into SMAD4 Loss in Pancreatic Cancer From Inducible Restoration of TGF-β Signaling.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer death in the United States. The TGF-β signaling protein SMAD family member 4 is lost in 60% of PDAC, and this has been associated with poorer prognosis. However, the mechanisms by which SMAD4 loss promotes PDAC development are not fully understood. We expressed SMAD4 in human PDAC cell lines BxPC3 and CFPAC1 by selection of stable clones containing an inducible SMAD4 tetracycline inducible expression system construct. After 24 hours of SMAD4 expression, TGF-β signaling-dependent G1 arrest was observed in BxPC3 cells with an increase in the G1 phase fraction from 48.9% to 71.5%. Inhibition of cyclin-dependent kinase inhibitor 1A by small interfering RNA eliminated the antiproliferative effect, indicating that up-regulation of cyclin-dependent kinase inhibitor 1A/p21 by TGF-β signaling is necessary for the phenotype. SMAD4 expression had no impact on invasion in BxPC3 cells, but reduced migration. Microarray analysis of gene expression at 8, 24, and 48 hours after SMAD4 expression characterized the regulatory impact of SMAD4 expression in a SMAD4-null PDAC cell line and identified novel targets of TGF-β signaling. Among the novel TGF-β targets identified are anthrax toxin receptor 2 (3.58× at 8 h), tubulin, β-3 class III (7.35× at 8 h), cell migration inducing protein, hyaluronan binding (8.07× at 8 h), IL-1 receptor-like 1 (0.403× at 8 h), regulator of G protein signaling 4 (0.293× at 8 h), and THAP domain containing 11 (0.262× at 8 h). The gene expression changes we observed upon restoration of TGF-β signaling provide numerous new targets for future investigations into PDAC biology and progression.

Anti-infective immunoadhesins from plants.

SummaryImmunoadhesins are recombinant proteins that combine the ligand‐binding region of a receptor or adhesion molecule with immunoglobulin constant domains. All FDA‐approved immunoadhesins are designed to modulate the interaction of a human receptor with its normal ligand, such as Etanercept (Enbrel®), which interferes with the binding of tumour necrosis factor (TNF) to the TNF‐alpha receptor and is used to treat inflammatory diseases such as rheumatoid arthritis. Like antibodies, immunoadhesins have long circulating half‐lives, are readily purified by affinity‐based methods and have the avidity advantages conferred by bivalency. Immunoadhesins that incorporate normal cellular receptors for viruses or bacterial toxins hold great, but as yet unrealized, potential for treating infectious disease. As decoy receptors, immunoadhesins have potential advantages over pathogen‐targeted monoclonal antibodies. Planet Biotechnology has specialized in developing anti‐infective immunoadhesins using plant expression systems. An immunoadhesin incorporating the cellular receptor for anthrax toxin, CMG2, potently blocks toxin activity in vitro and protects animals against inhalational anthrax. An immunoadhesin based on the receptor for human rhinovirus, ICAM‐1, potently blocks infection of human cells by one of the major causes of the common cold. An immunoadhesin targeting the MERS coronavirus is in an early stage of development. We describe here the unique challenges involved in designing and developing immunoadhesins targeting infectious diseases in the hope of inspiring further research into this promising class of drugs.

The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore.

Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited the function of the protective antigen (PA) pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA) domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.

Recent Developments in Anti-dotes Against Anthrax.

The etiologic agent of disease anthrax, Bacillus anthracis, causes recurrent outbreaks among the livestock and intermittent infections in humans across the world. Controlling animal infections by vaccination can minimize the incidence of disease in humans. Prevention of anthrax in occupationally exposed personnel is achieved through vaccination with either live spores or precipitates of culture supernatants from attenuated strains of B. anthracis. However, anthrax vaccination of the large human population is impractical as well as inappropriate. Broad-range antibiotics like amoxicillin, ciprofloxacin, clindamycin, streptomycin, and penicillin G are recommended for the treatment of human anthrax infections, but the threat of antibiotic resistant strains always remains. Moreover, in the absence of any specific symptom (s) during early infection, the diagnosis of anthrax is delayed causing elevated levels of anthrax toxin component which could be fatal. For these reasons, there is a need to develop new antimicrobial agents against virulent B. anthracis to effectively combat this fatal pathogen. Over the last two decades, extensive studies have been carried out to develop specific inhibitors against virulence factors of B. anthracis such as capsule, protective antigen, lethal factor and edema factor. Research has also been focused in developing inhibitors of anthrax toxin receptors (including the use of receptor decoys) and host furin endoproteases which are required for activation of toxin. This review highlights the recent progress made in developing the diverse countermeasures for anthrax infections targeting B. anthracis virulence factors and their counterparts in host.

Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8

ANTXR 1 and 2, also known as TEM8 and CMG2, are two type I membrane proteins, which have been extensively studied for their role as anthrax toxin receptors, but with a still elusive physiological function. Here we have analyzed the importance of N-glycosylation on folding, trafficking and ligand binding of these closely related proteins. We find that TEM8 has a stringent dependence on N-glycosylation. The presence of at least one glycan on each of its two extracellular domains, the vWA and Ig-like domains, is indeed necessary for efficient trafficking to the cell surface. In the absence of any N-linked glycans, TEM8 fails to fold correctly and is recognized by the ER quality control machinery. Expression of N-glycosylation mutants reveals that CMG2 is less vulnerable to sugar loss. The absence of N-linked glycans in one of the extracellular domains indeed has little impact on folding, trafficking or receptor function of the wild type protein expressed in tissue culture cells. N-glycans do, however, seem required in primary fibroblasts from human patients. Here, the presence of N-linked sugars increases the tolerance to mutations in cmg2 causing the rare genetic disease Hyaline Fibromatosis Syndrome. It thus appears that CMG2 glycosylation provides a buffer towards genetic variation by promoting folding of the protein in the ER lumen.

Regulatory mechanisms of anthrax toxin receptor 1-dependent vascular and connective tissue homeostasis

It is well known that angiogenesis is linked to fibrotic processes in fibroproliferative diseases, but insights into pathophysiological processes are limited, due to lack of understanding of molecular mechanisms controlling endothelial and fibroblastic homeostasis. We demonstrate here that the matrix receptor anthrax toxin receptor 1 (ANTXR1), also known as tumor endothelial marker 8 (TEM8), is an essential component of these mechanisms. Loss of TEM8 function in mice causes reduced synthesis of endothelial basement membrane components and hyperproliferative and leaky blood vessels in skin. In addition, endothelial cell alterations in mutants are almost identical to those of endothelial cells in infantile hemangioma lesions, including activated VEGF receptor signaling in endothelial cells, increased expression of the downstream targets VEGF and CXCL12, and increased numbers of macrophages and mast cells. In contrast, loss of TEM8 in fibroblasts leads to increased rates of synthesis of fiber-forming collagens, resulting in progressive fibrosis in skin and other organs. Compromised interactions between TEM8-deficient endothelial and fibroblastic cells cause dramatic reduction in the activity of the matrix-degrading enzyme MMP2. In addition to insights into mechanisms of connective tissue homeostasis, our data provide molecular explanations for vascular and connective tissue abnormalities in GAPO syndrome, caused by loss-of-function mutations in ANTXR1. Furthermore, the loss of MMP2 activity suggests that fibrotic skin abnormalities in GAPO syndrome are, in part, the consequence of pathophysiological mechanisms underlying syndromes (NAO, Torg and Winchester) with multicentric skin nodulosis and osteolysis caused by homozygous loss-of-function mutations in MMP2.

Infantile Systemic Hyalinosis Complicated with Right Atrial Thrombus and Pericardial Effusion in an Infant

Infantile systemic hyalinosis (ISH) is a rare multisystem fatal autosomal recessive disorder that involves widespread deposition of hyaline on connective tissues and certain internal organs. The major manifestations include painful articular contractures, hyperpigmentation, subcutaneous nodules, gingival hypertrophy, failure to thrive secondary to protein-losing enteropathy, and osteolytic bone lesions. In this paper, we report a 12-month-old girl with ISH presenting with recurrent diarrhea, failure to thrive, and refractory infections. A molecular study identified a homozygous missense mutation, c.134T>C; p.L45P, in exon 1 of the anthrax toxin receptor 2 (ANTRX2) gene. Our patient passed through an eventful course that included septic shock, central line infections, right atrial thrombosis, and pericardial effusion. She incurred acute bronchiolitis due to respiratory syncytial virus infection, which led to her death. In conclusion, this case report highlights that severe and life-threatening morbidities and complications can be encountered in ISH, to which some management options can be applied.