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Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly-Asp-containing peptide
S L Hart1, A M Knight, R P Harbottle
1Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, London, United Kingdom.
Abstract:
Ligands that bind mammalian cell surface integrins with high affinity can mediate cellular internalization. We show that particles of the bacteriophage fd that display the cyclic integrin-binding peptide sequence GGCRGDMFGC in a proportion of their major coat protein subunits bind to cells and are efficiently internalized. In the displayed peptide the conformation of the RGD motif is restricted within a hairpin loop formed by a disulfide bridge between the 2 cysteine residues. Cellular internalization of phage was demonstrated by confocal and non-confocal immunofluorescence microscopy of tissue-cultured cells incubated with phage particles. The phage were contained in juxtanuclear vesicles in the same serial sections as transferrin receptor but were not colocalized with the cell surface marker alkaline phosphatase. Cell binding and internalization was inhibited by preincubation of cells with the integrin-binding peptide GRGDSP, whereas the control peptide GRGESP had no inhibitory effect. These results indicate that cyclic integrin-binding peptides can be used to target and enter cells and that it should be possible to exploit such peptides for the introduction of DNA, drugs, or other macromolecules.
Insights
Bacteriophage fd particles displaying cyclic RGD peptides bind to cells and are internalized. This integrin-mediated process shows potential for delivering DNA, drugs, or macromolecules into cells.
Area of Science:
- Biotechnology
- Molecular Biology
- Cell Biology
Background:
- High-affinity ligands targeting mammalian cell surface integrins can facilitate cellular internalization.
- Bacteriophage display technology offers a platform for presenting peptides on viral surfaces.
Purpose of the Study:
- To investigate the use of bacteriophage fd displaying cyclic RGD peptides for cell targeting and internalization.
- To demonstrate the potential of engineered bacteriophages as vehicles for intracellular delivery.
Main Methods:
- Engineering bacteriophage fd to display cyclic RGD peptides (GGCRGDMFGC) on major coat protein subunits.
- Utilizing confocal and immunofluorescence microscopy to visualize phage-cell interactions and internalization.
- Employing peptide inhibition assays with GRGDSP and GRGESP to confirm integrin-mediated binding.
Main Results:
- Phage particles displaying cyclic RGD peptides were efficiently internalized by cultured cells.
- Internalized phage were localized in juxtanuclear vesicles, colocalizing with transferrin receptor but not alkaline phosphatase.
- Cell binding and internalization were specifically inhibited by the integrin-binding peptide GRGDSP.
Conclusions:
- Cyclic integrin-binding peptides displayed on bacteriophages can effectively target and enter mammalian cells.
- This phage-based system holds promise for the development of novel drug and gene delivery vectors.
- The RGD motif's conformation, stabilized by a disulfide bridge, is crucial for integrin recognition and internalization.