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The Journal of biological chemistry  2007;282(13):9834-9845.
Anthrax toxin, a three-component protein toxin secreted by Bacillus anthracis, assembles into toxic complexes at the surface of receptor-bearing eukaryotic cells. The protective antigen (PA) protein binds to receptors, either tumor endothelial cell marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), and orchestrates the delivery of the lethal and edema factors into the cytosol. TEM8 is reported to be over-expressed during tumor angiogenesis, whereas CMG2 is more widely expressed in normal tissues. To extend prior work on targeting of tumor with modified anthrax toxins, we used phage display to select PA variants that preferentially bind to TEM8 as compared to CMG2. Substitutions were randomly introduced into residues 605-729 of PA, within the C-terminal domain 4 of PA, which is the principal region that contacts receptor. Candidates were characterized in cellular cytotoxicity assays with CHO cells expressing either TEM8 or CMG2. A PA mutant having the substitutions R659S and M662R had enhanced specificity toward TEM8 over-expressing CHO cells. This PA variant also displayed broad and potent tumoricidal activity to various human tumor cells, especially to HeLa and A549/ATCC cells. By contrast, the substitution N657Q significantly reduced toxicity to TEM8 but not CMG2 over-expressing CHO cells. Our results indicate that certain amino acid substitutions within PA domain 4 create anthrax toxins that selectively kill human tumor cells. The PA R659S/M662R protein may be useful as a therapeutic agent for cancer treatment.
PMCID: PMC2530824  PMID: 17251181
2.  Capillary morphogenesis protein-2 is required for mouse parturition by maintaining uterine collagen homeostasis 
Capillary morphogenesis protein-2 (CMG2) functions as an anthrax toxin receptor that plays an essential role in anthrax pathogenesis. Although mutations in CMG2 have been identified to cause two human autosomal recessive disorders, Juvenile Hyaline Fibromatosis and Infantile Systemic Hyalinosis, both characterized by excess hyaline material deposition in connective tissues, the physiologic function of CMG2 remains elusive. To study the roles of CMG2 in normal physiology, here we performed detailed histological analyses of the CMG2-null mice we generated previously. While no morphological or histological defects were observed in CMG2−/− male mice, CMG2−/− female mice were unable to produce any offspring due to a defect in parturition. We found that deletion of CMG2 resulted in a diffuse deposition of collagen within the myometrium of CMG2−/− females, causing remarkable morphological changes to their uteri. This collagen accumulation also led to loss of smooth muscle cells in the myometrium of CMG2−/− mice, apparently disabling uterine contractile function during parturition. As a consequence, even though pregnant CMG2−/− mice were able to carry the gestation to full term, they were unable to deliver pups. However, the fully-developed fetuses could be successfully delivered by Cesarean section and survived to adulthood when fostered. Our results demonstrate that CMG2 is not required for normal mouse embryonic development but is indispensable for murine parturition. In parallel to its role in anthrax toxin binding and internalization, herein we provide evidence that CMG2 may function as a collagen receptor which is essential for maintaining collagen homeostasis in the uterus.
PMCID: PMC3376708  PMID: 22575514
Anthrax; anthrax toxin receptor; collagen; capillary morphogenesis protein-2; parturition
3.  Characterization of the interaction between anthrax toxin and its cellular receptors 
Cellular microbiology  2007;9(4):977-987.
Mutations in capillary morphogenesis gene 2 (CMG2), one of the two closely related proteins that act as anthrax toxin receptors, cause two rare human autosomal recessive conditions, juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH). Here we demonstrate that CMG2 proteins with certain JHF- and ISH-associated single amino acid substitutions in their von Willebrand factor A domain or transmembrane region do not function as anthrax toxin receptors. However, an ISH-associated CMG2 variant having a truncated cytosolic domain does still function as an anthrax receptor, and in fact makes cells hyper-sensitive to toxin, distinguishing the roles of CMG2 in physiology and anthrax pathology. Site-specific mutagenesis was used to characterize the role that domain 2 of the anthrax toxin protective antigen (PA) plays in interaction with CMG2, focusing on the interaction between the PA 2β3−2β4 loop and a pocket (Glu-122 pocket) adjacent to the metal ion-dependent adhesion site in CMG2. Substitutions that disrupted the salt bridge between PA Arg-344 and CMG2 Glu-122 decreased the affinity of PA to CMG2 3∼4-fold. Furthermore, mutation of CMG2 Tyr-119 (within the Glu-122 pocket) to His lowered the pH threshold for PA prepore-to-pore conversion in the endocytic pathway.
PMCID: PMC2459336  PMID: 17381430
4.  Recombinant Anthrax Toxin Receptor-Fc Fusion Proteins Produced in Plants Protect Rabbits against Inhalational Anthrax ▿ †  
Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.
PMCID: PMC3019684  PMID: 20956592
5.  Anthrax Toxin Receptor 2 is expressed in murine and tumor vasculature and functions in endothelial proliferation and morphogenesis 
Oncogene  2009;29(6):789-801.
The Capillary Morphogenesis Gene 2 (CMG2) gene encodes an Anthrax toxin receptor (ANTXR2) but the normal physiological function is not known. ANTXR2/CMG2 was originally identified as a result of up-regulation during capillary morphogenesis of endothelial cells cultured in vitro. We explored the hypothesis that key steps of the angiogenic process are either dependent or are influenced by ANTXR2/CMG2 activity. We describe the expression pattern of ANTXR2/CMG2 in several murine tissues and in normal breast and breast tumors. Endothelial expression was found in all of the tissues analyzed, in cultured endothelial cells and in breast tumor vessels; however ANTXR2/CMG2 expression was not restricted to this cell type. To assess potential angiogenic function, we utilized RNA interference to achieve significant reduction of ANTXR2/CMG2 expression in cultured human umbilical venous endothelial cells. Reduced ANTXR2/CMG2 expression resulted in significant inhibition of proliferation and reduced capacity of endothelial cells to form capillary-like networks in vitro, while overexpression of ANTXR2/CMG2 in HUVEC increased proliferation and capillary-like network formation. Little change in migration of endothelial cells was observed upon knockdown or overexpression. We conclude that ANTXR2/CMG2 functions to promote endothelial proliferation and morphogenesis during sprouting angiogenesis, consistent with the endothelial expression of ANTXR2/CMG2 in several vascular beds.
PMCID: PMC2939496  PMID: 19901963
ANTXR2; CMG2; angiogenesis; endothelial cells
6.  A FRET-Based High Throughput Screening Assay to Identify Inhibitors of Anthrax Protective Antigen Binding to Capillary Morphogenesis Gene 2 Protein 
PLoS ONE  2012;7(6):e39911.
Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein–protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.
PMCID: PMC3386954  PMID: 22768167
7.  Crystal Structure of the Engineered Neutralizing Antibody M18 Complexed to Domain 4 of the Anthrax Protective Antigen 
Journal of molecular biology  2009;387(3):680-693.
The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity single chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here we report the high resolution X-ray structures of three high affinity single chain antibodies in the 14B7 family; 14B7 and two high affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 Å resolution. These structures provide insights into the mechanism of neutralization and on the effect of various mutations on antibody affinity and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.
PMCID: PMC2670345  PMID: 19361425
8.  East African cassava mosaic-like viruses from Africa to Indian ocean islands: molecular diversity, evolutionary history and geographical dissemination of a bipartite begomovirus 
Cassava (Manihot esculenta) is a major food source for over 200 million sub-Saharan Africans. Unfortunately, its cultivation is severely hampered by cassava mosaic disease (CMD). Caused by a complex of bipartite cassava mosaic geminiviruses (CMG) species (Family: Geminivirideae; Genus: Begomovirus) CMD has been widely described throughout Africa and it is apparent that CMG's are expanding their geographical distribution. Determining where and when CMG movements have occurred could help curtail its spread and reveal the ecological and anthropic factors associated with similar viral invasions. We applied Bayesian phylogeographic inference and recombination analyses to available and newly described CMG sequences to reconstruct a plausible history of CMG diversification and migration between Africa and South West Indian Ocean (SWIO) islands.
The isolation and analysis of 114 DNA-A and 41 DNA-B sequences demonstrated the presence of three CMG species circulating in the Comoros and Seychelles archipelagos (East African cassava mosaic virus, EACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV). Phylogeographic analyses suggest that CMG’s presence on these SWIO islands is probably the result of at least four independent introduction events from mainland Africa occurring between 1988 and 2009. Amongst the islands of the Comoros archipelago, two major migration pathways were inferred: One from Grande Comore to Mohéli and the second from Mayotte to Anjouan. While only two recombination events characteristic of SWIO islands isolates were identified, numerous re-assortments events were detected between EACMV and EACMKV, which seem to almost freely interchange their genome components.
Rapid and extensive virus spread within the SWIO islands was demonstrated for three CMG complex species. Strong evolutionary or ecological interaction between CMG species may explain both their propensity to exchange components and the absence of recombination with non-CMG begomoviruses. Our results suggest an important role of anthropic factors in CMGs spread as the principal axes of viral migration correspond with major routes of human movement and commercial trade. Finer-scale temporal analyses of CMGs to precisely scale the relative contributions of human and insect transmission to their movement dynamics will require further extensive sampling in the SWIO region.
PMCID: PMC3560262  PMID: 23186303
Cassava; Begomoviruses; Dissemination; Africa; Phylogeography; Recombination
9.  Crystallization and preliminary X-ray analysis of the vWA domain of human anthrax toxin receptor 1 
The vWA domain of human anthrax toxin receptor 1 was overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 1.8 Å resolution.
The Gram-positive spore-forming bacterium Bacillus anthracis causes anthrax by secreting anthrax toxin, which consists of protective antigen (PA), lethal factor and oedema factor. Binding of PA to receptors triggers the multi-step process of anthrax toxin entry into target cells. Two distinct cellular receptors, ANTXR1 (also known as tumour endothelial marker 8; TEM8) and ANTXR2 (also known as capillary morphogenesis protein 2; CMG2), for anthrax toxin have been identified. Although the crystal structure of the extracellular von Willebrand factor A (vWA) domain of CMG2 has been reported, the difference between the vWA domains of TEM8 and CMG2 remains unclear because there are no structural data for the TEM8 vWA domain. In this report, the TEM8 vWA domain was expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Å resolution from a single crystal, which belonged to space group P1 with unit-cell parameters a = 65.9, b = 66.1, c = 74.4 Å, α = 63.7, β = 88.2, γ = 59.9°.
PMCID: PMC3079974  PMID: 21206026
human anthrax toxin receptor 1; von Willibrand factor A domain; tumour endothelial marker 8
10.  The Receptors that Mediate the Direct Lethality of Anthrax Toxin  
Toxins  2012;5(1):1-8.
Tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) are the two well-characterized anthrax toxin receptors, each containing a von Willebrand factor A (vWA) domain responsible for anthrax protective antigen (PA) binding. Recently, a cell-based analysis was used to implicate another vWA domain-containing protein, integrin β1 as a third anthrax toxin receptor. To explore whether proteins other than TEM8 and CMG2 function as anthrax toxin receptors in vivo, we challenged mice lacking TEM8 and/or CMG2. Specifically, we used as an effector protein the fusion protein FP59, a fusion between the PA-binding domain of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas aeruginosa exotoxin A. FP59 is at least 50-fold more potent than LF in the presence of PA, with 2 μg PA + 2 μg FP59 being sufficient to kill a mouse. While TEM8−/− and wild type control mice succumbed to a 5 μg PA + 5 μg FP59 challenge, CMG2−/− mice were completely resistant to this dose, confirming that CMG2 is the major anthrax toxin receptor in vivo. To detect whether any toxic effects are mediated by TEM8 or other putative receptors such as integrin β1, CMG2−/−/TEM8−/− mice were challenged with as many as five doses of 50 μg PA + 50 μg FP59. Strikingly, the CMG2−/−/TEM8−/− mice were completely resistant to the 5-dose challenge. These results strongly suggest that TEM8 is the only minor anthrax toxin receptor mediating direct lethality in vivo and that other proteins implicated as receptors do not play this role.
PMCID: PMC3564063  PMID: 23271637
anthrax; CMG2; FP59; integrin β1; Tem8
11.  DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast 
Molecular Biology of the Cell  2012;23(16):3240-3253.
DNA Pol ε synthesizes the leading strands, following the CMG (Cdc45/Mcm2-7/GINS) helicase, although the N-terminal polymerase domain of the catalytic subunit, Cdc20 in fission yeast, is dispensable for viability. We show that the C-terminal domain of Cdc20 plays the noncatalytic essential roles in both the assembly and progression of CMG helicase.
DNA polymerase epsilon (Pol ε) synthesizes the leading strands, following the CMG (Cdc45, Mcm2-7, and GINS [Go-Ichi-Nii-San]) helicase that translocates on the leading-strand template at eukaryotic replication forks. Although Pol ε is essential for the viability of fission and budding yeasts, the N-terminal polymerase domain of the catalytic subunit, Cdc20/Pol2, is dispensable for viability, leaving the following question: what is the essential role(s) of Pol ε? In this study, we investigated the essential roles of Pol ε using a temperature-sensitive mutant and a recently developed protein-depletion (off-aid) system in fission yeast. In cdc20-ct1 cells carrying mutations in the C-terminal domain of Cdc20, the CMG components, RPA, Pol α, and Pol δ were loaded onto replication origins, but Cdc45 did not translocate from the origins, suggesting that Pol ε is required for CMG helicase progression. In contrast, depletion of Cdc20 abolished the loading of GINS and Cdc45 onto origins, indicating that Pol ε is essential for assembly of the CMG complex. These results demonstrate that Pol ε plays essential roles in both the assembly and progression of CMG helicase.
PMCID: PMC3418317  PMID: 22718908
12.  Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein 
Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.
PMCID: PMC3179146  PMID: 21954339
molecular farming; transient; gene silencing suppressors; Nicotiana benthamiana; anthrax; fusion protein
13.  DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome 
eLife  2014;3:e03273.
The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.
eLife digest
Before a cell divides, it must duplicate its DNA so that each new cell inherits its own copy of the genome. To do this, the DNA double helix must be unwound so that the two individual strands of DNA can serve as templates for making new DNA molecules. Unwinding begins when two helicase complexes, termed the Mcm2-7 rings, are loaded together onto the DNA.
At first, the two Mcm2-7 rings encircle the double-stranded DNA and remain bound together in an inactive form. Activating the Mcm2-7 rings requires the binding of five other proteins to each ring, which forms two larger complexes called CMG helicases. When the CMG helicases form, the two DNA strands separate and an individual Mcm2-7 ring ends up encircling each of the single DNA strands. However, how an activated CMG complex is assembled, and how it binds to and unwinds DNA, is not fully understood.
Now, Costa et al. have determined the three-dimensional structure of the fruit fly CMG helicase bound to a DNA double helix with a single-stranded overhang at one end. The activated Mcm2-7 ring binds to the overhang, which confirms previous findings indicating that the activated helicase prefers single-stranded over double-stranded DNA. The structure also shows that, as a CMG helicase slides along the single-stranded DNA towards the double-stranded DNA, it is the ring complex's ‘motor domains’ that lead the way, while its DNA-binding domains trail behind.
Costa et al. also found that disrupting some of the interactions between two of the five proteins that bind to the Mcm2-7 ring either prevented the replicative helicase from forming or made it unstable. Furthermore, it was revealed that one of these two proteins—called Cdc45—was ideally placed to capture the strand of DNA that might be accidentally released from the Mcm2-7 ring. It was also discovered that when the complex is bound to DNA, the motor domains of the Mcm2-7 complex change shape from a flat ring to a spiral structure; the DNA-binding domains, however, remain in a flat ring. Costa et al. note that this structure is similar to that adopted by many viral and bacterial helicases, and that it even shares many features with the molecular machinery that breaks down unneeded or damaged proteins inside cells.
Finally, Costa et al. were able to image a structure composed of two CMG complexes bound together. This reveals the relative orientation of the two Mcm2-7 rings before they separate and move in opposite directions to unravel the DNA. The findings of Costa et al., combined with previous structural work in this field, demonstrate that the Mcm2-7 helicase complex can adopt many different shapes as it is assembled on DNA and activated to support DNA replication.
PMCID: PMC4359367  PMID: 25117490
DNA replication; Mcm2-7; helicase; motor proteins; replication fork; AAA+ ATPase; D. melanogaster
14.  Improving the Anti-Toxin Abilities of the CMG2-Fc Fusion Protein with the Aid of Computational Design 
PLoS ONE  2014;9(8):e104674.
CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.
PMCID: PMC4125234  PMID: 25101992
15.  1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) Inhibits Angiogenesis via Inhibition of CMG2 
Journal of medicinal chemistry  2013;56(5):1940-1945.
CMG2 is a transmembrane extracellular matrix binding protein that is also an anthrax toxin receptor. We have shown that high affinity CMG2 binders can inhibit angiogenesis and tumor growth. We recently described a high throughput FRET assay to identify CMG2 inhibitors. We now report the serendipitous discovery that PGG (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose) is a CMG2 inhibitor with anti-angiogenic activity. PGG is a gallotannin produced by a variety of medicinal plants that exhibits a wide variety of anti-tumor and other activities. We find that PGG inhibits CMG2 with a submicromolar IC50 and it also inhibits the migration of human dermal microvascular endothelial cells at similar concentrations in vitro. Finally, oral or intraperitoneal administration of PGG inhibits angiogenesis in the mouse corneal micropocket assay in vivo. Together, these results suggest that a portion of the in vivo anti-tumor activity of PGG may be the result of antiangiogenic activity mediated by inhibition of CMG2.
PMCID: PMC3600088  PMID: 23394144
pentagalloyl glucose; 5GG; angiogenesis; corneal neovascularization; cancer; polyphenol
16.  Anthrax Toxin Uptake by Primary Immune Cells as Determined with a Lethal Factor-β-Lactamase Fusion Protein 
PLoS ONE  2009;4(11):e7946.
To initiate infection, Bacillus anthracis needs to overcome the host innate immune system. Anthrax toxin, a major virulence factor of B. anthracis, impairs both the innate and adaptive immune systems and is important in the establishment of anthrax infections.
Methodology/Principal Findings
To measure the ability of anthrax toxin to target immune cells, studies were performed using a fusion of the anthrax toxin lethal factor (LF) N-terminal domain (LFn, aa 1–254) with β-lactamase (LFnBLA). This protein reports on the ability of the anthrax toxin protective antigen (PA) to mediate LF delivery into cells. Primary immune cells prepared from mouse spleens were used in conjunction with flow cytometry to assess cleavage and resulting FRET disruption of a fluorescent β-lactamase substrate, CCF2/AM. In spleen cell suspensions, the macrophages, dendritic cells, and B cells showed about 75% FRET disruption of CCF2/AM due to cleavage by the PA–delivered LFnBLA. LFnBLA delivery into CD4+ and CD8+ T cells was lower, with 40% FRET disruption. When the analyses were done on purified samples of individual cell types, similar results were obtained, with T cells again having lower LFnBLA delivery than macrophages, dendritic cells, and B cells. Relative expression levels of the toxin receptors CMG2 and TEM8 on these cells were determined by real-time PCR. Expression of CMG2 was about 1.5-fold higher in CD8+ cells than in CD4+ and B cells, and 2.5-fold higher than in macrophages.
Anthrax toxin entry and activity differs among immune cells. Macrophages, dendritic cells, and B cells displayed higher LFnBLA activity than CD4+ and CD8+ T cells in both spleen cell suspension and the purified samples of individual cell types. Expression of anthrax toxin receptor CMG2 is higher in CD4+ and CD8+ T cells, which is not correlated to the intracellular LFnBLA activity.
PMCID: PMC2775957  PMID: 19956758
17.  Expression of cmg1, an Exo-β-1,3-Glucanase Gene from Coniothyrium minitans, Increases during Sclerotial Parasitism 
During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains β-1,3-glucan as its major component. A PCR-based strategy was used to clone a β-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-β-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiae INVSc1 expressing the cmg1 gene secreted a ∼100-kDa β-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth of S. sclerotiorum by 35 and 85% at concentrations of 300 and 600 μg ml−1, respectively. A single copy of the cmg1 gene is present in the genome of C. minitans. Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum.
PMCID: PMC92660  PMID: 11157256
18.  Tumor Endothelium Marker-8 Based Decoys Exhibit Superiority over Capillary Morphogenesis Protein-2 Based Decoys as Anthrax Toxin Inhibitors 
PLoS ONE  2011;6(6):e20646.
Anthrax toxin is the major virulence factor produced by Bacillus anthracis. The toxin consists of three protein subunits: protective antigen (PA), lethal factor, and edema factor. Inhibition of PA binding to its receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) can effectively block anthrax intoxication, which is particularly valuable when the toxin has already been overproduced at the late stage of anthrax infection, thus rendering antibiotics ineffectual. Receptor-like agonists, such as the mammalian cell-expressed von Willebrand factor type A (vWA) domain of CMG2 (sCMG2), have demonstrated potency against the anthrax toxin. However, the soluble vWA domain of TEM8 (sTEM8) was ruled out as an anthrax toxin inhibitor candidate due to its inferior affinity to PA. In the present study, we report that L56A, a PA-binding-affinity-elevated mutant of sTEM8, could inhibit anthrax intoxication as effectively as sCMG2 in Fisher 344 rats. Additionally, pharmacokinetics showed that L56A and sTEM8 exhibit advantages over sCMG2 with better lung-targeting and longer plasma retention time, which may contribute to their enhanced protective ability in vivo. Our results suggest that receptor decoys based on TEM8 are promising anthrax toxin inhibitors and, together with the pharmacokinetic studies in this report, may contribute to the development of novel anthrax drugs.
PMCID: PMC3107238  PMID: 21674060
19.  Capillary morphogenesis gene 2 regulates adhesion and invasiveness of prostate cancer cells 
Oncology Letters  2014;7(6):2149-2153.
Capillary morphogenesis gene 2 (CMG2), also known as anthrax toxin receptor 2, has been indicated in the formation of new vasculature and in the internalisation of the anthrax toxin. Anti-angiogenesis therapy that targets this molecule has been investigated. However, our recent studies of this molecule have indicated that this gene may also play certain roles in cancer cells. The present study aimed to examine the expression of CMG2 in prostate cancer tissues and cell lines, and also its impact on cellular functions. The expression of CMG2 was detectable in normal and prostate cancer tissues. The prostate cancer cell lines appeared to have relatively high expression compared with the prostatic epithelial cells. Knockdown of CMG2 impaired the adherence of the prostate cancer cells. CMG2 overexpression resulted in decreasing invasiveness, while the knockdown of CMG2 contrastingly enhanced this ability. The altered expression of CMG2 in the prostate cancer cells did not affect the in vitro or in vivo growth of the cells. Taken together, these results show that CMG2 is expressed in prostatic epithelia and cancer cells. In addition to its role in the angiogenesis and the internalisation of anthrax toxin, CMG2 also plays an important role in regulating the adhesion and invasion of prostate cancer cells.
PMCID: PMC4049711  PMID: 24932305
capillary morphogenesis gene 2; anthrax toxin receptor 2; prostate cancer; adhesion; invasion
20.  The Structure of Tumor Endothelial Marker 8 (TEM8) Extracellular Domain and Implications for Its Receptor Function for Recognizing Anthrax Toxin 
PLoS ONE  2010;5(6):e11203.
Anthrax toxin, which is released from the Gram-positive bacterium Bacillus anthracis, is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds a receptor on the surface of the target cell and further assembles into a homo-heptameric pore through which EF and LF translocate into the cytosol. Two distinct cellular receptors for anthrax toxin, TEM8/ANTXR1 and CMG2/ANTXR2, have been identified, and it is known that their extracellular domains bind PA with low and high affinities, respectively. Here, we report the crystal structure of the TEM8 extracellular vWA domain at 1.7 Å resolution. The overall structure has a typical integrin fold and is similar to that of the previously published CMG2 structure. In addition, using structure-based mutagenesis, we demonstrate that the putative interface region of TEM8 with PA (consisting of residues 56, 57, and 154–160) is responsible for the PA-binding affinity differences between the two receptors. In particular, Leu56 was shown to be a key factor for the lower affinity of TEM8 towards PA compared with CMG2. Because of its high affinity for PA and low expression in normal tissues, an isolated extracellular vWA domain of the L56A TEM8 variant may serve as a potent antitoxin and a potential therapeutic treatment for anthrax infection. Moreover, as TEM8 is often over-expressed in tumor cells, our TEM8 crystal structure may provide new insights into how to design PA mutants that preferentially target tumor cells.
PMCID: PMC2887854  PMID: 20585457
21.  Potential implications of Helicobacter pylori-related neutrophil-activating protein 
Helicobacter pylori (H. pylori) virulence factors promote the release of various chemoattractants/inflammatory mediators, including mainly the neutrophil-attractant chemokine interleukin-8 and neutrophil-activating protein (NAP), involved in H. pylori-induced gastric pathologies. Co-administration of Chios mastic gum (CMG), which inhibits H. pylori NAP, with an H. pylori eradication regimen might add clinical benefits against H. pylori-related gastric pathologies, but possibly not CMG as main therapy. Although H. pylori NAP and other H. pylori-related cytotoxins [i.e., vaculating cytotoxin (VacA)] appear to play a major role in generating and maintaining the H. pylori-associated gastric inflammatory response and H. pylori NAP is a promising vaccine candidate against H. pylori infection (H. pylori-I), concerns regarding its potential drawbacks, particularly neurogenic ones, due to possible cross-mimicry, should be considered. Possible cross-mimicry between H. pylori NAP and/or bacterial aquaporin (AQP) and neural tissues may be associated with the anti-AQP-4 antibody-related neural damage in multiple sclerosis (MS)/neuromyelitis optica patients. Moreover, the sequence homology found between H. pylori VacA and human Na+/K+-ATPase A subunit suggests that antibodies to VacA involve ion channels in abaxonal Schwann cell plasmalemma resulting in demyelination in some patients. A series of factors have been implicated in inducing blood-brain barrier (BBB) disruption, including inflammatory mediators (e.g., cytokines and chemokines induced by H. pylori-I) and oxidative stress. BBB disruption permits access of AQP4-specific antibodies and T lymphocytes to the central nervous system, thereby playing a major role in multiple sclerosis pathogenesis. Relative studies show a strong association between H. pylori-I and MS. H. pylori-I induces humoral and cellular immune responses that, owing to the sharing of homologous epitopes (molecular mimicry), cross-react with components of nerves, thereby contributing and perpetuating neural tissue damage. Finally, H. pylori NAP also plays a possible pathogenetic role in both gastric and colon oncogenesis.
PMCID: PMC3270508  PMID: 22346256
Helicobacter pylori; Neutrophil-activating protein; Chios mastic gum; Cross-mimicry; Multiple sclerosis; Demyelination; Gastric carcinogenesis
22.  The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo 
Journal of Bacteriology  1998;180(7):1947-1950.
A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels of Bradyrhizobium japonicum holocytochrome c550 expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.
PMCID: PMC107112  PMID: 9537397
23.  104 Immunogenicity and Safety Aspects of Adeno-Associated Virus–Like Particles (AAVLPS) as Carriers for B-Cell Vaccines 
The World Allergy Organization Journal  2012;5(Suppl 2):S51-S52.
Adeno-associated viruses (AAV) are non-human pathogenic and replication defective ssDNA viruses. The surface of AAV consists of 60 capsomers, which can be exploited for high density display of recombinant peptides. AAV-like particles (AAVLP) can be generated via assembly of the recombinant capsid protein VP3. The aim of this study was to evaluate the uptake mechanism, immunogenicity and safety aspects of an AAVLP-displayed B-cell epitope, taking ovalbumin (OVA) as a model antigen/allergen.
An OVA derived linear B-cell epitope and for control purposes OVA-non related peptide TP18 (cholesterol-ester transfer protein 18) were inserted into capsid protein VP3 of AAVLPs.
Life cell microscopy indicated that AAVLP internalized into HeLa epithelial cells and remained in intracellular vesicles up to 18 hours. When we immunized BALB/c subcutaneously, sera of AAVLP-OVA immunized mice showed similar titres of OVA-specific IgG1 compared to mice immunized with OVA protein. However, in OVA immunized mice high OVA-specific IgE levels could be recorded, whereas immunizations with OVA-AAVLP rendered background IgE levels only. In accordance, sera of OVA mice which permitted mast cell degranulation upon OVA trigger in a specific β-hexosaminidase release assay, whereas sera of OVA-AAVLP mice did not contain anaphylactogenic antibodies. In an in vivo anaphylaxis experiment, upon intravenous OVA challenge OVA-immunized mice presented significant drop of body temperature, whereas AAVLP-OVA mice remained unaffected.
Our study demonstrates the immunogenicity, safety and efficacy of AAVLP as display system of B-cell epitopes for vaccination.
PMCID: PMC3512624
24.  Targeting the anthrax receptors, TEM-8 and CMG-2, for anti-angiogenic therapy 
The anthrax toxin receptors tumor endothelial marker-8 (TEM-8) and capillary morphogenesis gene-2 (CMG-2) are responsible for allowing entry of anthrax toxin into host cells. However, these receptors were first discovered due to their enhanced expression on endothelial cells undergoing blood vessel growth or angiogenesis in in vitro or in vivo model systems. Targeting and inhibiting angiogenesis is an important strategy for current anti-cancer therapies and treatment of retinal diseases. Structures, tissue expression, and interactions of the TEM-8 and CMG-2 proteins have been documented, and functional roles for these receptors in angiogenesis have recently emerged. TEM-8 appears to regulate endothelial cell migration and tubule formation whereas a role for CMG-2 in endothelial proliferation has been documented. TEM-8 and CMG-2 bind differentially to extracellular matrix proteins including collagen I, collagen IV and laminin and these properties may be responsible for their apparent roles in regulating endothelial cell behavior during angiogenesis. TEM-8-binding moieties have also been suggested to be useful in selectively targeting anti-angiogenic and anti-tumorigenic therapies to tumor endothelium. Additionally, studies of modified forms of lethal toxin (LeTx) have demonstrated that targeted inhibition of MAPKs within tumor vessels may represent an efficacious anti-angiogenic strategy.
PMCID: PMC3066103  PMID: 21196249
Endothelial; angiogenesis; anthrax; intracellular signaling; extracellular matrix
25.  Fabrication and characterization of nuclear localization signal-conjugated glycol chitosan micelles for improving the nuclear delivery of doxorubicin 
Supramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS)-modified polymeric micelles were studied with the aim of improving nuclear drug delivery.
In this research, cholesterol-modified glycol chitosan (CHGC) was synthesized. NLS-conjugated CHGC (NCHGC) was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa and HepG2 cells.
The degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC) micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater cellular uptake and higher amounts of DOX in the nuclei of HeLa cells than free DOX and DOX-loaded CHGC (DCHGC) micelles. The half maximal inhibitory concentration (IC50) values of free DOX, DCHGC, and DNCHGC micelles against HepG2 cells were 4.063, 0.591, and 0.171 μg/mL, respectively. Moreover, the IC50 values of free DOX (3.210 μg/mL) and the DCHGC micelles (1.413 μg/mL) against HeLa cells were nearly 6.96- and 3.07-fold (P < 0.01), respectively, higher than the IC50 value of the DNCHGC micelles (0.461 μg/mL).
The results of this study suggest that novel NCHGC micelles could be a potential carrier for nucleus-targeting delivery.
PMCID: PMC3459689  PMID: 23049255
polymeric micelles; drug delivery; nucleus-targeting delivery

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