Expression and purification of functional human anthrax toxin receptor (ATR/TEM8) binding domain from Escherichia coli

Ding, Zhiping; Bradley, Kenneth A.; Arnaout, M. Amin; Xiong, Jian-Ping

doi:10.1016/j.pep.2006.04.011

Cited by 10 publications

(8 citation statements)

References 39 publications

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“…Thus, glycosylation of TEM8, in particular at position 262, is required for efficient PA binding. Recombinant, bacterial expressed, TEM8 vWA domain is fully competent for PA binding [ 51 ], thus glycosylation per se is not required for ligand binding. Therefore the absence of PA binding to the 3NA mutant is likely due to a defect in folding in the cellular context in the absence of glycosylation, which would be consistent with the full ER retention of TEM8 3NA.…”

Section: Resultsmentioning

confidence: 99%

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

2015

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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.

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Section: Resultsmentioning

confidence: 99%

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

2015

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“…Recent studies have revealed the previously unsuspected biological role of LRP6 for prophylaxis and treatment of anthrax toxicity. LRP6-specific antibodies have protected cell cultures from destruction by LT, indicating that this newly identified co-receptor might be a viable target in providing protection against the effects of accumulated LT …”

Section: The Medical “Arsenal” Against Anthrax: Vaccines Antibodies A...mentioning

confidence: 99%

Current and Future Medical Approaches To Combat the Anthrax Threat

Bouzianas

2010

J. Med. Chem.

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“…The protein expression and purification were performed following previously reported procedures [6]. Briefly, the pGEX4T-1 plasmid was transformed to TOP10 chemically competent E. coli (Invitrogen), and selected single colonies were shaken in 1 l lysogeny broth medium containing 100 μg/ml ampicillin at 37°C and 250 rpm.…”

Section: Methodsmentioning

confidence: 99%

“…It shares the extracellular von Willebrand factor type A (vWA) domain with the other receptor for anthrax toxin called capillary morphogenesis protein 2 (CMG2/ANTXR2) [6, 7]. TEM8 is reported to be selectively overexpressed on both tumor endothelial and cancer cells during tumor angiogenesis, whereas CMG2 is more widely expressed in normal tissues [8].…”

Section: Introductionmentioning

confidence: 99%

Imaging tumor endothelial marker 8 using an 18F-labeled peptide

Quan

Yang

Gao

et al. 2011

Eur J Nucl Med Mol Imaging

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Purpose Tumor endothelial marker 8 (TEM8) has been reported to be upregulated in both tumor cells and tumor-associated endothelial cells in several cancer types. TEM8 antagonists and TEM8-targeted delivery of toxins have been developed as effective cancer therapeutics. The ability to image TEM8 expression would be of use in evaluating TEM8-targeted cancer therapy. Methods A 13-meric peptide, KYNDRLPLYISNP (QQM), identified from the small loop in domain IV of protective antigen of anthrax toxin was evaluated for TEM8 binding and labeled with 18F for small-animal PET imaging in both UM-SCC1 head-and-neck cancer and MDA-MB-435 melanoma models. Results A modified ELISA showed that QQM peptide bound specifically to the extracellular vWA domain of TEM8 with an IC50 value of 304 nM. Coupling 4-nitrophenyl 2-18F-fluoropropionate with QQM gave almost quantitative yield and a high specific activity (79.2±7.4 TBq/mmol, n=5) of 18F-FP-QQM at the end of synthesis. 18F-FP-QQM showed predominantly renal clearance and had significantly higher accumulation in TEM8 high-expressing UM-SCC1 tumors (2.96±0.84 %ID/g at 1 h after injection) than TEM8 low-expressing MDA-MB-435 tumors (1.38±0.56 %ID/g at 1 h after injection). Conclusion QQM peptide bound specifically to the extracellular domain of TEM8. 18F-FP-QQM peptide tracer would be a promising lead compound for measuring TEM8 expression. Further efforts to improve the affinity and specificity of the tracer and to increase its metabolic stability are warranted.

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Expression and purification of functional human anthrax toxin receptor (ATR/TEM8) binding domain from Escherichia coli

Cited by 10 publications

References 39 publications

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

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

Current and Future Medical Approaches To Combat the Anthrax Threat

Imaging tumor endothelial marker 8 using an 18F-labeled peptide

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