Phage display screening of epithelial cell monolayers treated with EGTA: identification of peptide FDFWITP that modulates tight junction activity.
Phage display was used to screen for peptides that modulate the activity of epithelial cell tight junctions. Panning with a phage library that displays random 7-mers was performed using monolayers of human bronchial epithelial cells (16HBE14o(-)) treated with a calcium chelator, ethylene glycol-bis(2-aminoethylether)- N, N, N', N'-tetraacetic acid (EGTA), to increase accessibility to the junctional complex/paracellular space, followed by subtractive panning. A novel peptide, FDFWITP, identified as a potential tight junction modulator, was synthesized in linear and cyclic forms with lysine residues added to improve solubility. The cyclic form of the peptide reduced transepithelial electrical resistance (TER) in a concentration-dependent manner (80% reduction at 100 microM and 95% reduction at 500 microM) and was reversible within 2 h; the linear form only affected TER at the highest concentration. Interestingly, the constrained peptide did not increase permeation of the model small molecule, fluorescein. The highly selective activity of FDFWITP supports the hypothesis that ions and small molecules may be transported paracellularly across tight junctions by separate pathways.
Herman
Richard E
RE
Nastech Pharmaceutical Company, Inc., Bothell, Washington 98021, USA.
Makienko
Ekaterina G
EG
Prieve
Mary G
MG
Fuller
Mark
M
Houston
Michael E
ME
Johnson
Paul H
PH
D000595:Amino Acid Sequence; D000818:Animals; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004285:Dogs; D004533:Egtazic Acid / Q000494:pharmacology*; D017097:Electric Impedance; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D010539:Permeability / Q000187:drug effects; D019108:Tight Junctions / Q000187:drug effects* / Q000502:physiology
D002614:Chelating Agents; D019151:Peptide Library; D010455:Peptides; D004533:Egtazic Acid; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium