The coiled-coil domain of occludin can act to organize structural and functional elements of the epithelial tight junction.
Occludin is an integral membrane protein that has been suggested to play a role in the organization and dynamic function of the epithelial tight junction (TJ). A number of other proteins have also been described to localize to the TJ. We have used a novel bait peptide method to investigate potential protein-protein interactions of the putative coiled-coil domain of occludin with some of these other TJ proteins. A 27-amino acid peptide of the human occludin sequence was synthesized, biotinylated at the N terminus, and modified to contain a photoactive moiety at either its hydrophobic or hydrophilic surface. These bait peptides were alpha-helical in solution, characteristic of coiled-coil structures. Photoactivation studies in the presence and absence of control peptides were used to assess the potential interactions in polarized sheets of a human intestinal cell line T84. Although a large number of proteins associated with the TJ or that are known to be involved in regulatory events of epithelial cells failed to be specifically labeled, occludin itself, ZO-1, protein kinase C-zeta, c-Yes, the regulatory subunit of phosphatidylinositol 3-kinase, and the gap junction component connexin 26 were specifically labeled. Our data demonstrate the potential of one specific domain of occludin, contained within 27 amino acids, to coordinate the binding of proteins that have been previously suggested to modulate TJ structure and function.
Nusrat
A
A
Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, USA.
Chen
J A
JA
Foley
C S
CS
Liang
T W
TW
Tom
J
J
Cromwell
M
M
Quan
C
C
Mrsny
R J
RJ
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000502:physiology* / Q000648:ultrastructure*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D062793:Occludin; D010449:Peptide Mapping / Q000379:methods; D017510:Protein Folding; D013329:Structure-Activity Relationship; D019108:Tight Junctions / Q000502:physiology*
D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin