In vitro protein complex formation with cytoskeleton-anchoring domain of occludin identified by limited proteolysis.
Occludin is an essential membrane protein component of cellular tight junctions, participating in both cell-cell adhesion in the paracellular space and anchoring of the junctional complex to the cytoskeleton. The latter function is accomplished through binding of the C-terminal cytoplasmic region to scaffolding proteins that mediate binding to cytoskeletal actin. We isolated a structural domain from both the bacterial-expressed C-terminal cytoplasmic region of human occludin and native cellular occludin, extracted from epithelial (Madin-Darby canine kidney) or endothelial (human brain) cells, by limited proteolysis with trypsin. This human occludin domain contains the last 119 amino acids as identified by N-terminal sequencing and peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Based on the sequence and secondary structure prediction, this domain contains 4 of 5 alpha-helices in the C-terminal region and is linked to the fourth membrane-spanning region by a loosely structured tethering polypeptide. Comparison of circular dichroism spectra of recombinant proteins corresponding to the entire C-terminal region versus only the binding domain region also supports the interpretation that the helical structural elements are concentrated in that domain. Co-immunoprecipitation of this domain with ZO-2 demonstrated preservation of the specificity of the scaffolding protein-binding function, and binding studies with immobilized ZO-2 suggest the presence of multiple ZO-2 binding sites in this domain. These results provide a basis for development of a structural model of the ZO-binding site that can be used to investigate regulation of tight junction anchoring by intracellular signaling events.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D003599:Cytoskeleton / Q000378:metabolism*; D017931:DNA Primers; D004285:Dogs; D006868:Hydrolysis; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D062793:Occludin; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D014357:Trypsin / Q000378:metabolism; D062827:Zonula Occludens-2 Protein
D017931:DNA Primers; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C569046:TJP2 protein, human; D062827:Zonula Occludens-2 Protein; D014357:Trypsin