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Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain
Published on: December 12, 2017
Preparation of a claudin-targeting molecule using a C-terminal fragment of Clostridium perfringens enterotoxin
Chiaki Ebihara1, Masuo Kondoh, Naoki Hasuike
1Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Machida-shi, Tokyo 194-8543, Japan.
Abstract:
Although most malignant tumors are epithelia-derived carcinomas, methods for specific and effective delivery of antitumor agents to carcinomas have not been developed. Recent reports indicate that epithelia overexpress claudin-3 and -4, which are integral membrane proteins of epithelial tight junctions. This suggests that claudins can be targeted for tumor therapy, but there is not currently a method for delivering drugs to claudin-expressing cells. In the present study, we evaluated whether a potent claudin-4-binding C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) would allow targeting to claudin-4-expressing cells. We fused C-CPE to the protein synthesis inhibitory factor (PSIF), which lacks the cell binding domain of Pseudomonas exotoxin. This fusion protein, C-CPE-PSIF, was cytotoxic to MCF-7 human breast cancer cells, which express endogenous claudin-4, but it was not toxic to mouse fibroblast L cells, which lack endogenous claudin-4. The cytotoxicity of C-CPE-PSIF was attenuated by pretreating the MCF-7 cells with C-CPE but not bovine serum albumin. Also, deletion of the claudin-4-binding region of C-CPE reduced the cytotoxicity of C-CPE-PSIF. Finally, we found that C-CPE-PSIF is toxic to L cells expressing claudin-4 but not to normal L cells or cells expressing claudin-1, -2, or -5. These results indicate that use of the C-CPE peptide may provide a novel way to target drugs to claudin-expressing cells.
Insights
A novel fusion protein targets claudin-4 expressing cancer cells. This C-CPE-PSIF conjugate shows specific toxicity to claudin-4 positive cells, offering a new strategy for targeted cancer therapy.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Carcinomas, the most common malignant tumors, lack specific drug delivery methods.
- Epithelial cells, including those in carcinomas, overexpress claudin-3 and claudin-4 tight junction proteins.
- Targeting claudins presents a potential therapeutic strategy, but effective delivery methods are needed.
Purpose of the Study:
- To investigate the potential of a claudin-4 binding fragment of Clostridium perfringens enterotoxin (C-CPE) for targeted drug delivery.
- To evaluate a fusion protein, C-CPE-PSIF, for its ability to selectively target and induce toxicity in claudin-4 expressing cells.
Main Methods:
- Constructed a fusion protein (C-CPE-PSIF) by linking C-CPE to protein synthesis inhibitory factor (PSIF).
- Assessed the cytotoxicity of C-CPE-PSIF against MCF-7 human breast cancer cells (claudin-4 positive) and mouse fibroblast L cells (claudin-4 negative).
- Investigated the specificity of C-CPE-PSIF by testing its toxicity on L cells engineered to express claudin-4 or other claudins (claudin-1, -2, -5).
Main Results:
- C-CPE-PSIF demonstrated significant cytotoxicity towards claudin-4 expressing MCF-7 cells.
- Toxicity was specific, as C-CPE-PSIF was not toxic to normal L cells lacking claudin-4.
- Pre-treatment with C-CPE, but not BSA, attenuated the cytotoxicity, confirming claudin-4 binding specificity.
- A mutated C-CPE lacking the binding region significantly reduced cytotoxicity.
- C-CPE-PSIF specifically killed claudin-4 expressing L cells, but not normal L cells or those expressing claudin-1, -2, or -5.
Conclusions:
- The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) can be utilized for targeted delivery to claudin-4 expressing cells.
- The C-CPE-PSIF fusion protein represents a novel therapeutic approach for targeting claudin-4 positive carcinomas.
- This strategy holds promise for developing specific and effective antitumor agents for epithelial-derived cancers.
