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

Jacquez, Pedro; Avila, Gustavo A.; Boone, Kyle; Agamyrat, Altiyev; Puschhof, Jens; Roland, Sauter; Arigi, Emma; Ruiz, Blanca; Peng, Xiuli; Almeida, Igor C.; Sherman, Michael B; Xiao, Chuan; Sun, Jianjun

doi:10.1371/journal.pone.0130832

Cited by 8 publications

(11 citation statements)

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“…NBD, an environmental sensitive dye, has little fluorescence in aqueous solution, but emits strong fluorescence when it inserts into lipid membranes. Thus, NBD has been used as a fluorescence marker for protein membrane insertion (41–43). Earlier, we have obtained evidence that the central Helix-Turn-Helix motif of EsxA inserts into the liposomal membrane at low pH (28).…”

Section: Resultsmentioning

confidence: 99%

Membrane Insertion ofMycobacterium tuberculosisEsxA in Cultured Lung Epithelial Cells

Zhang

Aguilera

Reyes

et al. 2020

Preprint

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10EsxA has long been recognized as an important virulence factor of Mycobacterium tuberculosis 11 (Mtb) that plays an essential role in Mtb cytosolic translocation presumably by penetrating 12 phagosomal membranes with its acidic pH-dependent membrane permeabilizing activity (MPA). 13 However, current data suggest that the observed cytolytic activity of EsxA at neutral pH is due to 14 contamination of ASB-14, a detergent used in EsxA protein purification, and the role of EsxA 15 MPA in Mtb cytosolic translocation is also questionable. Here, we have obtained evidence that it 16 is ASB-14, not EsxA that causes cytolysis at neutral pH. Quantitative liquid chromatography and 17 mass spectrometry showed that even after gel filtration, dialysis, or passing through detergent 18 removal column, the remaining ASB-14 in the EsxA protein solution was still at a concentration 19 enough to kill cultured lung epithelial cells. When treated with trypsin or proteinase K, the digested 20 EsxA protein solution with ASB-14 was still cytotoxic. Interestingly, however, we have found that 21 the exogenously added EsxA is endocytosed into lung epithelial cells and inserts into the host 22 membranes within acidic subcellular compartments, which can be blocked by cytochalasin D and 23 bafilomycin A. It is for the first time EsxA is found to insert into the host membranes within acidic 24 subcellular compartments. 25 insertion 27 Importance: 28 EsxA has long been recognized as an important virulence factor of Mycobacterium tuberculosis 29 (Mtb) that plays an essential role in Mtb virulence. However, current data regarding to its role in 30 Mtb virulence are controversial. Here, we have obtained evidence showing that the cytolytic 31 activity of EsxA at neutral pH is due to contamination of ASB-14, a detergent used in EsxA 32 preparation. Moreover, it is for the first time we have found that EsxA protein is endocytosed into 33 lung epithelial cells and inserts into the host membranes within acidic subcellular compartments, 34 implicating an important role of the acidic pH-dependent membrane permeabilizing activity of 35 EsxA in Mtb virulence.36 37 38 39 2 Introduction 40 Mycobacterium tuberculosis (Mtb), the causative bacterial pathogen for tuberculosis disease, 41 infects one-third of the world's population and causes over one million deaths worldwide each 42year (1, 2). It is estimated that an airborne droplet carrying 1-3 Mtb bacilli is enough to cause 43 systemic dissemination and infection (3, 4). When Mtb is inhaled into the lung, it is engulfed into 44 alveolar macrophage. Instead of being destroyed in phagolysosomes like many other intracellular 45 pathogens, Mtb develops effective strategies to evade host defense mechanisms. Recent studies 46 have shown that Mtb is able to rupture the phagosomal membrane and translocate to the cytosol, 47 where it replicates, eventually lyses the macrophage and spreads to the surrounding non-48 phagocytic cells, such as alveolar endothelial and epithelial cells (5-10). Thus, cytosolic ...

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Section: Resultsmentioning

confidence: 99%

Membrane Insertion ofMycobacterium tuberculosisEsxA in Cultured Lung Epithelial Cells

Zhang

Aguilera

Reyes

et al. 2020

Preprint

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“…In Origami B cells, both glutathione reductase and thioredoxin reductase are deleted so that the cytosol becomes favorable for disulfide bond formation [ 44 ]. Site-directed mutagenesis and mass spectrometry of the recombinant protein demonstrate that the two disulfide bonds in the Ig domain are formed by C255–C279 and C230–C315, respectively ( Figure 3 C) [ 45 ]. While a high-resolution structure of the Ig domain is still not available, computational modeled structures and the low-resolution EM maps have revealed valuable structural details ( Figure 4 A).…”

Section: The Ig-like Domain Of Antxr2mentioning

confidence: 99%

“…While a high-resolution structure of the Ig domain is still not available, computational modeled structures and the low-resolution EM maps have revealed valuable structural details ( Figure 4 A). Two modeled structures from two different groups show high similarity [ 45 , 46 ]. Moreover, a low-resolution EM map (~14 Å) of the PA-ectodomain heptameric complex was determined by single-particle 3D reconstruction of negatively stained samples ( Figure 4 B,C).…”

Section: The Ig-like Domain Of Antxr2mentioning

confidence: 99%

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

Sun¹,

Jacquez²

2016

Toxins

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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.

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“…HFS is caused by either a homozygous or compound heterozygous mutation in the anthrax toxin receptor 2 (ANTXR2) gene, additionally termed the capillary morphogenesis protein 2 gene, which is localized on chromosome 4q21 (1,2). ANTXR2 encodes for a type I transmembrane (TM) protein, which consists of an extracellular N terminal von Willebrand A (vWA) domain, an immunoglobulin-like domain, a TM domain and a cytosolic tail (3)(4)(5)(6). TM is able to bind to laminin and collagen IV via its vWA domain, and thus may regulate basement membrane matrix assembly (7).…”

Section: Introductionmentioning

confidence: 99%

Two novel mutations in the ANTXR2 gene in a Chinese patient suffering from hyaline fibromatosis syndrome: A case report

et al. 2018

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Hyaline fibromatosis syndrome (HFS; MIM 228600) is a rare autosomal recessive disorder characterized by the abnormal growth of hyalinized fibrous tissue at subcutaneous regions on the scalp, ears and neck. The disease is caused by either a homozygous or compound heterozygous mutation of the anthrax toxin receptor 2 (ANTXR2) gene. The present study describes a patient with HFS confirmed by clinical examination as well as histopathological and genetic analyses. Numerous painless and variable‑sized subcutaneous nodules were observed on the scalp, ear, trunk and four extremities of the patient. With increasing age, the number and size of the nodules gradually increased in the patient. The patient additionally presented with severe gingival thickening and developed pearly papules on the ears, back and penis foreskin. Biopsies of ear nodules revealed that the tumor was located in the dermis, and no marked alterations were observed in the epidermis compared with healthy patients. Spindle‑shaped or round tumor cells were revealed to be immersed in the eosinophilic hyaline ground substance. Furthermore, a skeletal X‑ray of the patient revealed multiple low‑density imaging on the right distal humerus. Compound heterozygous mutations in the ANTXR2 gene were identified in the patient: c.470_472del in exon 5 and c.1073 delC in exon 13. c.470_472del were revealed to be inherited from his mother and father, respectively. These two mutations, c.470_472del and c.1073 delC, to the best of our knowledge, have not previously been identified. Identification of the mutations in ANTXR2 may make prenatal diagnosis of HFS possible during future pregnancies.

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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

Cited by 8 publications

References 61 publications

Membrane Insertion ofMycobacterium tuberculosisEsxA in Cultured Lung Epithelial Cells

Membrane Insertion ofMycobacterium tuberculosisEsxA in Cultured Lung Epithelial Cells

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

Two novel mutations in the ANTXR2 gene in a Chinese patient suffering from hyaline fibromatosis syndrome: A case report

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