Research Article
No access
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


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.

Get full access to this article

View all available purchase options and get full access to this article.


Bal MS, Castro V, Piontek J, Rueckert C, Walter JK, Shymanets A, Kurig B, Haase H, Nurnberg B, and Blasig IE. The hinge region of the scaffolding protein of cell contacts, zonula occludens protein 1, regulates interacting with various signaling proteins. J Cell Biochem 113: 934–945, 2012.
Balda MS, Garrett MD, and Matter K. The ZO-1-associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density. J Cell Biol 160: 423–432, 2003.
Blasig IE, Bellmann C, Cording J, del Vecchio G, Zwanziger D, Huber O, and Haseloff RF. Occludin protein family: oxidative stress and reducing conditions. Antioxid Redox Signal 15: 1195–1219, 2011.
Blasig IE, Winkler L, Lassowski B, Mueller SL, Zuleger N, Krause E, Krause G, Gast K, Kolbe M, and Piontek J. On the self-association potential of transmembrane tight junction proteins. Cell Mol Life Sci 63: 505–514, 2006.
Brast S, Grabner A, Sucic S, Sitte HH, Hermann E, Pavenstädt H, Schlatter E, and Giarimboli G. The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization. FASEB J 26: 976–986, 2012.
Brown RC and Davis TP. Calcium modulation of adherens and tight junction function. a potential mechanism for blood–brain barrier disruption after stroke. Stroke 33: 1706–1711, 2002.
Brown RC and Davis TP. Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells. Biochem Biophys Res Commun 327: 1114–1123, 2005.
Caraballo JC, Yshii C, Butti ML, Westphal W, Borcherding JA, Allamargot C, and Comellas AP. Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway. Am J Physiol Lung Cell Mol Physiol 300: L569–L578, 2011.
Cording J, Berg J, Käding N, Bellmann C, Westphal JK, Milatz S, Günzel D, Wolburg H, Piontek J, Huber O, and Blasig IE. Tight junctions: claudins regulate the interactions between occludin, tricellulin and marvelD3, which, inversely, modulate claudin oligomerization. J Cell Sci 162: 554–564, 2012.
Dörfel MJ and Huber O. Modulation of tight junction structure and function by kinases and phosphatases targeting occludin. J Biomed Biotechnol 2012: 807356, 2012.
Dukes JD, Paul Whitley P, and Chalmers AD. The PIKfyve inhibitor YM201636 blocks the continuous recycling of the tight junction proteins claudin-1 and claudin-2 in MDCK cells. PLoS One 7: e28659, 2012.
Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, and Tsukita S. Occludin-A novel integral membrane-protein localizing at tight junctions J Cell Biol 123: 1777–1788, 1993.
Gaddie KJ and Kirley TL. Conserved polar residues stabilize transmembrane domains and promote oligomerization in human nucleoside triphosphate diphosphohydrolase 3. Biochemistry 48: 9437–9447, 2009.
Gonzalez-Mariscal L, Betanzos A, and Avila-Flores A. MAGUK proteins: structure and role in the tight junction. Semin Cell Dev Biol 11: 315–324, 2000.
Gonzalez-Mariscal L, Hernandez S, and Vega J. Inventions designed to enhance drug delivery across epithelial and endothelial cells through tha paracellular pathway. Recent Pat Drug Deliv Formul 2: 145–176, 2008.
Haseloff RF, Krause E, Bigl M, Mikoteit K, Stanimirovic D, and Blasig IE. Differential protein expression in brain capillary endothelial cells induced by hypoxia and posthypoxic reoxygenation. Proteomics 6: 1803–1809, 2006.
Klausner RD, Donaldson JG, and Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffick and organelle structure. J Cell Biol 116: 1071–1080, 1992.
Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J, and Blasig IE. Structure and function of claudins. Biochim Biophys Acta 1778: 631–645, 2008.
Lochhead JJ, McCaffrey G, Quigley CE, Finch J, DeMarco KM, Nametz N, and Davis TP. Oxidative stress increases blood–brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation. J Cereb Blood Flow Metab 30: 1625–1636, 2010.
Mark KS and Davis TP. Cerebral microvascular changes inpermeability and tight junctions induced by hypoxia/reoxygenation. Am J Physiol Heart Circ Physiol 282: H1485–H1494, 2002.
McCaffrey G, Seelbach MJ, Staatz WD, Nametz N, Quigley C, Campos CR, Brooks TA, and Davis TP. Occludin oligomeric assembly at tight junctions of the blood–brain barrier is disrupted by peripheral inflammatory hyperalgesia. J Neurochem 106: 2395–2409, 2008.
McCaffrey G, Willis CL, Staatz WD, Nametz N, Quigley CA, Hom S, Lochhead JJ, and Davis TP. Occludin oligomeric assemblies at tight junctions of the blood–brain barrier are altered by hypoxia and reoxygenation stress. J Neurochem 110: 58–71, 2009.
Morgan B, Sobotta MC, and Dick TP. Measuring EGSH and H2O2 with roGFP2-based redox probes. Free Radic Biol Med 51: 1943–1951, 2011.
Müller SL, Portwich M, Schmidt A, Utepbergenov DI, Huber O, Blasig IE, and Krause G. The tight junction protein occludin and the adherens junction protein alpha-catenin share a common interaction mechanism with ZO-1. J Biol Chem 280: 3747–3756, 2005.
Pelis RM, Dangprapai Y, Cheng Y, Zhang X, Terpstra J, and Wright SH. Functional significance of conserved cysteines in the human organic cation transporter 2. Am J Physiol Renal Physiol 303: F313–F320, 2012.
Piontek J, Fritzsche S, Cording J, Richter S, Hartwig J, Walter M, Yu D, Turner JR, Gehring C, Rahn H-P, Wolburg H, and Blasig IE. Elucidating the principles of the molecular organization of heteropolymeric tight junction strands. Cell Mol Life Sci 68: 3903–3918, 2011.
Piontek J, Winkler L, Wolburg H, Muller SL, Zuleger N, Piehl C, Wiesner B, Krause G, and Blasig IE. Formation of tight junction: determinants of homophilic interaction between classic claudins. FASEB J 22: 146–158, 2008.
Rajasekaran SA, Barwe SP, Gopal J, Ryazantsev S, Schneeberger EE, and Rajasekaran AK. Na-K-ATPase regulates tight junction permeability through occludin phosphorylation in pancreatic epithelial cells. Am J Physiol Gastrointest Liver Physiol 292: G124–G133, 2007.
Rentea RM, Liedel JL, Welak SR, Cassidy LD, Mayer AN, Pritchard KA Jr, Oldham KT, and Gourlay DM. Intestinal alkaline phosphatase administration in newborns is protective of gut barrier function in a neonatal necrotizing enterocolitis rat model. J Pediatr Surg 47: 1135–1142, 2012.
Roy A, Kucukural A, and Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5: 725–738, 2010.
Shen L, Weber CR, Raleigh DR, Yu D, and Turner JR. Tight junction pore and leak pathways: a dynamic duo. Annu Rev Physiol 73: 283–309, 2011.
Storjohann L, Holst B, and Schwartz TW. A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39. Biochemistry 47: 9198–9207, 2008.
Walter JK, Rueckert C, Voss M, Mueller SL, Piontek J, Gast K, and Blasig IE. The oligomerization of the coiled coil-domain of occluddin is redox-sensitive. Ann N Y Acad Sci 1165: 19–27, 2009.
Wang P, Wu Y, Li X, Ma X, and Zhong L. Thioredoxin and thioredoxin reductase control tissue factor activity by thiol redox-dependent mechanism. J Biol Chem 288: 3346–3358, 2013.
Wolburg H, Liebner S, and Lippoldt A. Freeze-fracture studies of cerebral endothelial tight junctions. Methods Mol Med 89: 51–66, 2003.
Yaffe Y, Shepshelovitch J, Nevo-Yassaf I, Yeheskel A, Shmerling H, Kwiatek JM, Gaus K, Pasmanik-Chor M, and Hirschberg K. The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia. J Cell Sci 125: 3545–3556, 2012.
Yguerabide J, Schmidt JA, and Yguerabide EE. Lateral mobility in membranes as detected by fluorescence recovery after photobleaching. Biophys J 40: 69–75, 1982.
Yu ASL, McCarthy KM, Francis SA, McCormack JM, Lai J, Rogers RA, Lynch RD, and Schneeberger EE. Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells. Am J Physiol Cell Physiol 288: C1231–C1241, 2005.
Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9: 40, 2008.

Information & Authors


Published In

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


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


Request permissions for this article.




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.


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.

Metrics & Citations



Export citation

Select the format you want to export the citations of this publication.

View Options

Get Access

Access content

To read the fulltext, please use one of the options below to sign in or purchase access.

Society Access

If you are a member of a society that has access to this content please log in via your society website and then return to this publication.

Restore your content access

Enter your email address to restore your content access:

Note: This functionality works only for purchases done as a guest. If you already have an account, log in to access the content to which you are entitled.

View options


View PDF/ePub

Full Text

View Full Text







Copy the content Link

Share on social media

Back to Top