1Departments of Molecular Pharmacology and Urology, 2Departments of Medicine and Pathology, and 3Departments of Cell Biology and Medicine, Albert Einstein College of Medicine, Bronx; 4Affinity BioReagents, Golden, Colorado; and 5Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York
Submitted 29 November 2004 ; accepted in final form 20 January 2005
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ABSTRACT |
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anthrax; epithelia; lung; skin; intestine; toxin entry; receptor; bacterial pathogenesis
In recent years, many important advances have been achieved in the anthrax field. These findings are expected to stimulate the development of new inhibitors against anthrax toxin (18, 23). The anthrax bacterium releases a toxin that is essential for lethal effects. This virulence factor is composed of three proteins: protective antigen (PA, which binds to cellular receptors), lethal factor (LF, which is a protease), and edema factor (EF, which belongs to the adenyl cyclase class of proteins). The crystal structures of PA, LF, and EF have been solved (12, 20, 21). LF and EF are individually nontoxic and require association with PA to enter the cytosol and to produce many of the symptoms of anthrax infection. The combination of PA and LF forms the lethal toxin, whereas the association of PA with EF forms the edema toxin. During cellular infection, PA binds anthrax toxin receptor (ATR) and is cleaved at the cell surface by furin and/or a furin-like protease. Cleaved PA oligomerizes and binds to EF or LF or both. The complex is then internalized via endocytosis (1, 2, 14) and trafficked to the acidic environment of endosomes, which promotes channel formation and translocation of LF or EF (10, 17).
The edema factor is an adenylate cyclase that induces a significant increase in the intracellular concentration of cAMP. Elevated levels of cAMP in the host cells alter water homeostasis, which leads to swelling and edema. The lethal factor is required for cell death and morbidity. LF is a protease that cleaves members of the mitogen-activated protein kinase (MAPK) kinase family, thereby disrupting three MAPK signaling pathways. The direct involvement of these pathways in LT death is unknown (17).
The identification of cellular receptors for anthrax toxin represents valuable progress in understanding the molecular events involved in the intoxication mechanism and in the development of antitoxins. The receptor ATR is a type I membrane protein and is expressed at moderately high levels on the cell surface (6). The extracellular region of ATR contains a von Willebrand factor type A (VWA) domain, which may modulate protein-protein interactions. This VWA domain constitutes the direct binding site for PA. In addition, a metal ion-dependent adhesion site motif, localized within the VWA domain, is important for toxin binding (57).
ATR is encoded by the tumor endothelial marker 8 (TEM8) gene. Three variants result from alternative splicing of the TEM8 gene. The long isoform (TEM8) is a transmembrane protein of 564 amino acids, with a long proline-rich cytoplasmic tail. The medium isoform (ATR) is a 368- amino acid protein, which possesses a short cytoplasmic tail and diverges from the long isoform at the last four amino acids at the COOH terminus. The long and medium isoforms are identical throughout the extracellular region, the putative transmembrane domain, and a portion of the cytoplasmic tail. Because they both contain the VWA domain that binds to PA, they both function as PA receptors (6). To the contrary, the short isoform lacks any sequence for membrane attachment. As a consequence, this putative secreted protein does not function as a PA receptor (22). Interestingly, ATR/TEM8 is highly conserved in different species, and mouse and human homologs share 98% sequence identity in the extracellular domain (6).
Although the physiological function of ATR/TEM8 is still unknown, multiple lines of evidence suggest a role in the regulation of angiogenesis. In fact, several independent investigators have shown that, in humans, TEM8 is preferentially expressed in endothelial cells within colonic tumors (9, 19, 24). In mice, TEM8 was shown to be highly expressed in tumor vessels, as well as in the vasculature, of developing embryos, but undetectable in normal tissues. Furthermore, a second protein was identified to function as an ATR: the human capillary morphogenesis protein 2 (CMG2) (4, 15, 25). Currently, CMG2 is the protein most similar to ATR/TEM8. CMG2 and ATR possess common characteristics, including a type I transmembrane domain and a VWA domain. These two proteins share 40% amino acid identity throughout their sequence and 60% identity within their VWA domain. Interestingly, similarly to ATR/TEM8, CMG2 was shown to bind PA with its VWA domain. CMG2 is expressed in a variety of tissues, but it is upregulated in human umbilical vein endothelial cells during the process of capillary formation. Taken together, these findings pinpoint that one key function of ATR/TEM8 may be the regulation of the neovasculature.
Before the molecular cloning of ATR, the PA binding receptor was tentatively localized to the basolateral surface of polarized epithelial cells. A series of studies demonstrated that EF enters epithelial cells through the basolateral but not the apical surface, suggesting that the receptor expression is restricted to the basolateral membrane of polarized epithelial cells. As such, the exact cellular distribution of ATR/TEM8 remains unknown (endothelial vs. epithelial). For example, the expression of ATR/TEM8 in epithelial cells has not been reported to date.
In the current study, we have examined the tissue-specific and cellular distribution of ATR/TEM8 in a variety of normal mouse and human tissues. For this purpose, we generated and characterized a novel polyclonal antibody that selectively recognizes an extracellular epitope within the VWA domain of the ATR/TEM8 protein. The generation of this novel antibody allowed us to identify the cellular targets of anthrax toxin at the molecular level.
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MATERIALS AND METHODS |
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Animal studies. This study was conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal protocols were preapproved by Institutional Animal Care and Use Committees. For all of these experiments, we used 3-mo-old male wild-type mice in the C57BL/6 background. The mice were housed and maintained in a barrier facility at the Albert Einstein College of Medicine. They were housed under 12:12-h light-dark cycle conditions and had ad libitum access to chow (Picolab 20; PMI Nutrition International) and water. After the mice were euthanized, the lungs were removed and insufflated with 2 ml of 10% neutrally buffered formalin. Small intestine samples and skin biopsies were surgically removed and fixed in 10% neutrally buffered formalin for 24 h, after which the samples were placed in 70% ethanol until processing. The tissue samples were paraffin embedded, and 4- to 5-µm-thick sections were cut and placed on Super-Frost Plus slides (Fisher) for immunohistochemistry (8).
Construction of ATR/TEM8 cDNA. The cDNAs encoding the long, medium, and short isoforms of ATR/TEM8 were reisolated and subcloned into TOPO, a mammalian expression vector (Invitrogen), according to the manufacturer's instructions. A Myc-tag epitope was placed at the COOH terminus of each construct. For isolation of the cDNA, we employed human Quick Clone cDNA (Clontech) and the Platinum Taq high-fidelity polymerase (GIBCO-BRL). Primers for amplification were designed based on the following three accession numbers: AF421380, NM032208, and BC012074. Also, a Kozak sequence was placed immediately ahead of the start site (GCCACCATG). The correctness of all clones was verified by DNA sequencing.
Cell culture and transfection. CHO cells were grown in RPMI supplemented with glutamine, antibiotics (penicillin and streptomycin), and 10% fetal calf serum. CHO cells were transiently transfected using the Lipofectamine transfection reagent (Invitrogen) as per the manufacturer's instructions, and cellular expression was analyzed 36 h posttransfection.
Antibody production. A polyclonal antibody to ATR/TEM8 was generated by immunization of New Zealand White rabbits with a synthetic peptide (residues 92107; LMKLTEDREQIRQGLEC) corresponding to a sequence in the extracellular domain of human ATR/TEM8, containing an exogenously added COOH-terminal cysteine residue to facilitate maleimide conjugation to keyhole limpet hemocyanin. The resulting IgGs were purified from serum by ammonium sulfate precipitation, followed by resuspension in PBS, and subjected to peptide affinity chromatography.
Western blot analysis. Cells were lysed in hot sample buffer (13). Samples were then collected and homogenized with the use of a 26-gauge needle and 1-ml syringe. Murine tissue lysates were mixed with an appropriate volume of sample buffer containing a reducing agent (520 mM DTT final concentration). Protein lysates were resolved by SDS-PAGE (10% acrylamide) under reducing conditions and transferred to nitrocellulose membranes (Schleicher and Schuell). The protein bands were visualized with Ponceau S (Sigma). Membranes were blocked with 4% nonfat dried milk in TBST (20 mM Tris·HCl, 150 mM NaCl, and 0.1% Tween 20) supplemented with 1% bovine serum albumin. Blots were then incubated at room temperature for 1 h with primary antibody diluted in TBST/1% bovine serum albumin. Horseradish peroxidase-conjugated secondary antibodies were used to visualize bound primary antibodies with the SuperSignal enhanced chemiluminescence substrate (Pierce).
Peptide competition. CHO cells were transiently transfected with cDNA encoding the long isoform and subjected to preparative SDS-PAGE gel. After transfer, the nitrocellulose membrane was cut into strips and incubated with anti-ATR/TEM8 polyclonal antibody alone or in combination with peptides. Horseradish peroxidase-conjugated secondary antibodies were used to visualize bound primary antibodies with the SuperSignal enhanced chemiluminescence substrate (Pierce).
Immunohistochemistry. Paraffin sections were deparaffinized in xylene (twice, 10 min each), hydrated through a graded series of ethanol washes and placed in PBS. Antigen retrieval was performed by heating at 95°C for 15 min in citrate buffer. Endogenous peroxide activity was quenched by incubation with the peroxidase blocking reagent (DAKO). Sections were incubated overnight with the anti-ATR/TEM8 polyclonal antibody. After being washed, the slides were incubated with a biotin-conjugated secondary antibody. The slides were then incubated with horseradish peroxidase-conjugated streptavidin (DAKO). Bound antibodies were visualized using diaminobenzidine as the substrate. The sections were then counterstained with hematoxylin and dehydrated. The slides were mounted with a xylene-based mounting medium (Micromount, Surgipath) and observed with an Olympus IX 70 inverted microscope.
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RESULTS |
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In this report, we attempt to elucidate the distribution of ATR/TEM8 in tissues that constitute the primary targets for anthrax infection. For this purpose, we generated and characterized a novel anti-ATR/TEM8 polyclonal antibody that selectively recognizes all three ATR/TEM8 isoforms (long, medium, and short).
Novel anti-ATR/TEM8 polyclonal antibody detects all three ATR/TEM8 isoforms. Figure 1A shows a schematic diagram of the three ATR/TEM8 isoforms resulting from alternative splicing of the TEM8 gene. The long isoform (TEM8) contains 564 amino acids with a transmembrane region and a long proline-rich cytoplasmic tail. The medium isoform (ATR) encodes a 368-amino acid protein with a transmembrane region and a short cytoplasmic tail. The long and medium isoforms are identical throughout the extracellular and transmembrane domains but diverge at the last four amino acids of the cytoplasmic tail at the COOH terminus. As such, the medium and long isoforms both function as receptors for the anthrax toxin (6). To the contrary, the short isoform encodes a 333-amino acid protein, which is identical to the other two isoforms in the extracellular domain but profoundly diverges just before the putative transmembrane domain and is lacking any sequence for membrane attachment. As a consequence, the short putative secreted isoform does not function as a receptor for the anthrax toxin.
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An alignment of the COOH-terminal domain sequences of the three isoforms illustrates that the three isoforms profoundly diverge at the COOH terminus (Fig. 1B). Note that the short isoform lacks a transmembrane domain, whereas the medium isoform has a 25-amino acid cytoplasmic tail, compared with the 221-amino acid cytoplasmic tail of the long isoform.
To test the immunoreactivity of our novel anti-ATR/TEM8 antibody, we transiently transfected CHO cells with Myc-tagged cDNA encoding the short, medium, and long ATR/TEM8 isoforms. Western blot analysis revealed that the ATR/TEM8 antibody successfully recognizes all three isoforms (Fig. 2A). Importantly, the different isoforms can be distinguished based on their respective molecular weights. The short isoform is a 45-kDa protein, the medium isoform is a 60-kDa protein, and the long isoform is an
80- to 85-kDa doublet. This doublet is probably due to multiple glycosylation events. Interestingly, this doublet was also detected in untransfected CHO cells, suggesting that the ATR/TEM8 antibody recognizes the long endogenous isoform. To independently verify these results, we used three isoforms containing a Myc tag at the COOH terminal and performed immunoblotting with an anti-Myc polyclonal antibody. Figure 2B shows that all three isoforms are strongly expressed in CHO cells. These results demonstrate the specificity and the strong affinity of the novel anti-ATR/TEM8 polyclonal antibody, as it detects exogenous as well as endogenous ATR/TEM8 isoforms.
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ATR/TEM8 is highly expressed in epithelial cells lining B. anthracis three sites of entry: lung, skin, and small intestine. Next, we attempted to evaluate the detailed tissue distribution of ATR/TEM8 by immunohistochemistry. We tested the ATR/TEM8 expression in the three tissues that represent the main routes of entry of the anthrax bacterium, i.e., lung, skin, and intestine. Because the pulmonary form is the most lethal form of the anthrax disease, we first assessed ATR/TEM8 expression on paraffin-embedded sections from mouse lung. Surprisingly, ATR/TEM8 expression was strongly detectable in the respiratory epithelium of the bronchi of the lung (Fig. 5). In particular, an intense staining was evident in the ciliated epithelial cells surrounding the luminal surface. In addition, ATR/TEM8 was also expressed in the smooth muscle cells surrounding the vessels, as well as in epithelial cells lining the alveoli. We observed a weak or undetectable staining in the endothelial cells lining the pulmonary vessels (Fig. 5). Taken together, the tissue distribution of ATR/TEM8, as determined by immunohistochemistry with the anti-ATR/TEM8 polyclonal antibody, correlated with the primary uptake sites of the anthrax bacterium.
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DISCUSSION |
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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