Highly conserved cysteines are involved in the oligomerization of occludin-redox dependency of the second extracellular loop.
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.


OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
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.
1 Leibniz-Institut für Molekulare Pharmakologie (FMP) , Berlin, Germany .
Christian
C
Bellmann
Sophie
S
Schreivogel
Ramona
R
Günther
Sebastian
S
Dabrowski
Michael
M
Schümann
Hartwig
H
Wolburg
Ingolf E
IE
Blasig
D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D062793:Occludin / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D019108:Tight Junctions / Q000378:metabolism
D062793:Occludin; D003545:Cysteine