In vitro protein complex formation with cytoskeleton-anchoring domain of occludin identified by limited proteolysis.

Bihung Peng, James Lee, Gerald Campbell

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)49644-49651
Number of pages8
JournalThe Journal of biological chemistry
Volume278
Issue number49
StatePublished - Dec 5 2003

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Proteolysis
Occludin
Cytoskeleton
Tight Junctions
Protein Binding
Proteins
Binding Sites
Peptides
Peptide Mapping
Structural Models
Cell adhesion
Circular Dichroism
Immunoprecipitation
Recombinant Proteins
Cell Adhesion
Trypsin
Ionization
Mass spectrometry
Canidae
Actins

ASJC Scopus subject areas

  • Biochemistry

Cite this

In vitro protein complex formation with cytoskeleton-anchoring domain of occludin identified by limited proteolysis. / Peng, Bihung; Lee, James; Campbell, Gerald.

In: The Journal of biological chemistry, Vol. 278, No. 49, 05.12.2003, p. 49644-49651.

Research output: Contribution to journalArticle

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