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© The Rockefeller University Press, 0021-9525/1997//399 $5.00
The Journal of Cell Biology, Volume 136, Number 2, , 1997 399-409

Article

A Synthetic Peptide Corresponding to the Extracellular Domain of Occludin Perturbs the Tight Junction Permeability Barrier



Vivian Wong*,{ddagger} and Barry M. Gumbiner*

* Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021; and {ddagger} Department of Physiology, University of California at San Francisco, San Francisco, California 94120

Occludin, the putative tight junction integral membrane protein, is an attractive candidate for a protein that forms the actual sealing element of the tight junction. To study the role of occludin in the formation of the tight junction seal, synthetic peptides (OCC1 and OCC2) corresponding to the two putative extracellular domains of occludin were assayed for their ability to alter tight junctions in Xenopus kidney epithelial cell line A6. Transepithelial electrical resistance and paracellular tracer flux measurements indicated that the second extracellular domain peptide (OCC2) reversibly disrupted the transepithelial permeability barrier at concentrations of < 5 µM. Despite the increased paracellular permeability, there were no changes in gross epithelial cell morphology as determined by scanning EM. The OCC2 peptide decreased the amount of occludin present at the tight junction, as assessed by indirect immunofluorescence, as well as decreased total cellular content of occludin, as assessed by Western blot analysis. Pulse-labeling and metabolic chase analysis suggested that this decrease in occludin level could be attributed to an increase in turnover of cellular occludin rather than a decrease in occludin synthesis. The effect on occludin was specific because other tight junction components, ZO-1, ZO-2, cingulin, and the adherens junction protein E-cadherin, were unaltered by OCC2 treatment. Therefore, the peptide corresponding to the second extracellular domain of occludin perturbs the tight junction permeability barrier in a very specific manner. The correlation between a decrease in occludin levels and the perturbation of the tight junction permeability barrier provides evidence for a role of occludin in the formation of the tight junction seal.

Abbreviations used in this paper: aa, amino acid; TER, transepithelial electrical resistance.

This work was partially supported by Cancer Center Support grant NCI-P30-CA-08748 and American Heart Association grant-in-aid 92006540. V. Wong was partially supported by National Institutes of Health predoctoral grants, NRSA-DK07265-130031 and NRSA-DK07265-130031, and a graduate opportunity fellowship from the University of California at San Francisco.

Address all correspondence to Barry M. Gumbiner, Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 564, New York 10021. Tel.: (212) 639-6146. Fax: (212) 717-3047.


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