Research Article
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Published Online: 10 February 2014

Highly Conserved Cysteines Are Involved in the Oligomerization of Occludin—Redox Dependency of the Second Extracellular Loop

Publication: Antioxidants & Redox Signaling
Volume 20, Issue Number 6

Abstract

The tight junction (TJ) marker occludin is a 4-transmembrane domain (TMD) protein with unclear physiological and pathological functions, interacting with other TJ proteins. It oligomerizes and is redox sensitive. However, oligomerization sites and mechanisms are unknown. Aims: To identify hypoxia-sensitive binding sites, we investigated the consequences of amino-acid substitutions of highly conserved cysteines in human occludin, under normal and hypoxic incubations. Results: (i) The extracellular loop 2 (ECL2) showed homophilic trans- and cis-association between opposing cells and along the cell membrane, respectively, caused by a loop properly folded via an intraloop disulfide bridge between the shielded C216 and C237. Hypoxia and reductants prevented the associations. (ii) C82 in TMD1 directly cis-associated without disulfide formation. (iii) C76 in TMD1 and C148 in TMD2 limited the trans-interaction; C76 also limited occludin-related paracellular tightness and changed the strand morphology of claudin-1. (iv) The diminished binding strength found after substituting C82, C216, or C237 was accompanied by increased occludin mobility in the cell membrane. Innovation: The data enable the first experimentally proven structural model of occludin and its homophilic interaction sites, in which the ECL2, via intraloop disulfide formation, has a central role in occludin's hypoxia-sensitive oligomerization and to regulate the structure of TJs. Conclusion: Our findings support the new concept that occludin acts as a hypoxiasensor and contributes toward regulating the TJ assembly redox dependently. This is of pathogenic relevance for tissue barrier injury with reducing conditions. The ECL2 disulfide might be a model for four TMD proteins in TJs with two conserved cysteines in an ECL. Antioxid. Redox Signal. 20, 855–867.

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Information & Authors

Information

Published In

cover image Antioxidants & Redox Signaling
Antioxidants & Redox Signaling
Volume 20Issue Number 6February 20, 2014
Pages: 855 - 867
PubMed: 23923978

History

Published in print: February 20, 2014
Published online: 10 February 2014
Published ahead of print: 17 September 2013
Published ahead of production: 7 August 2013
Accepted: 6 August 2013
Revision received: 18 July 2013
Received: 4 March 2013

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Christian Bellmann
Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.
Sophie Schreivogel
Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.
Ramona Günther
Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.
Sebastian Dabrowski
Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.
Michael Schümann
Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.
Hartwig Wolburg
Institut für Pathologie und Neuropathologie, Universität Tübingen, Tübingen, Germany.
Ingolf E. Blasig
Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.

Notes

Address correspondence to:Dr. Ingolf E. BlasigLeibniz-Institut für Molekulare Pharmakologie (FMP)Robert-Rössle-Str. 10Berlin-Buch 13125Germany
E-mail: [email protected]

Author Disclosure Statement

No competing financial interests exist.

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