Abstract
We investigated whether a synthetic tetrameric branched peptide based on the conserved TFLK motif from mammary-associated serum amyloid A3 (M-SAA3) is more efficient than the monomeric peptide at up-regulating MUC3 expression and examined the possible mechanism(s) and biological significance of this process. We used standard solid-phase methods to synthesize a tetrameric branched peptide (sequence GWLTFLKAAG) containing a trilysine core, termed the TFLK-containing 10-mer BP. The aberrant expression of transcription factors was analyzed using a transcription factor protein/DNA array. MUC3 and relevant transcription factors were detected using real-time PCR and/or Western blots. The luciferase assay, EMSA, and ChIP assays were used to analyze the activity of the human MUC3 promoter. The bacterial adherence assay was used to evaluate the in vitro inhibition of enteropathogenic Escherichia coli or enterohemorrhage E. coli serotype O157:H7 (EHEC O157:H7) adherence to HT-29-Gal cells after treatment with the TFLK-containing 10-mer BP. In HT-29-Gal cells, the TFLK-containing 10-mer BP induced higher levels of MUC3 expression than the M-SAA3-derived N-terminal 10-mer monomeric peptide, and MUC3 expression was activated through transcriptional mechanisms, including the induction of multiple transcription factors and further binding with their cis-elements between nucleotides -242 and -62 within MUC3 promoter. Interestingly, the TFLK-containing 10-mer BP dramatically inhibited enteropathogenic E. coli and EHEC O157:H7 adherence to the HT-29-Gal cells compared with the controls. This finding suggests a potential therapeutic use for this peptide to prevent gastrointestinal infection.
Highlights
A tetrameric branched peptide (BP) containing a TFLK motif was designed to induce MUC3 expression and inhibit bacteria adherence
We investigated whether a synthetic tetrameric branched peptide based on the conserved TFLK motif from mammaryassociated serum amyloid A3 (M-SAA3) is more efficient than the monomeric peptide at up-regulating MUC3 expression and examined the possible mechanism(s) and biological significance of this process
TFLK-containing 10-Mer-BP Enhances MUC3 Expression in HT-29-Gal Cells—To determine the ideal concentration of the TFLK-containing 10-mer BP for the activation of MUC3 expression in the HT-29-Gal cells, we analyzed the MUC3 mRNA levels by real-time PCR in the HT-29-Gal cells stimulated with different concentrations of the TFLK-containing 10-mer BP (50, 100, 200, 400, and 800 g/ml) and compared it to the untreated HT-29-Gal cells at three time points (0.5, 1, and 3 h)
Summary
A tetrameric branched peptide (BP) containing a TFLK motif was designed to induce MUC3 expression and inhibit bacteria adherence. Increasing the peptide size increases the difficulty of identifying peptides with high affinity and selectivity; multimeric peptides comprising simple branched structures and a short arm may offer an optimal compromise between molecular size and the ease of preparation, yield, and purity of the final product This strategy of combinatorial chemistry prompted us to explore whether the multimeric branched peptide based on the conserved TFLK motif from M-SAA3 induces increased expression of MUC3 in intestinal epithelial cells and exerted a more significant effect. In this study we demonstrate that compared with the M-SAA3-derived N-terminal 10-mer monomeric peptide containing the TFLK motif (residues 1–10, sequence GWLTFLKAAG), the TFLK-containing 10-mer BP stimulates higher levels of human MUC3 expression in intestinal epithelial cells and dramatically inhibits the adherence of EPEC and EHEC O157: H7. We explored the mechanism(s) of transcriptional regulation of human MUC3 mucin by the TFLK-containing 10-mer BP in HT-29-Gal cells
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