Multiple protein interactions involving proposed extracellular loop domains of the tight junction protein occludin - PubMed (original) (raw)

Multiple protein interactions involving proposed extracellular loop domains of the tight junction protein occludin

Asma Nusrat et al. Mol Biol Cell. 2005 Apr.

Abstract

Occludin is a tetraspan integral membrane protein in epithelial and endothelial tight junction (TJ) structures that is projected to have two extracellular loops. We have used peptides emulating central regions of human occludin's first and second loops, termed O-A:101-121 and O-B:210-228, respectively, to examine potential molecular interactions between these two regions of occludin and other TJ proteins. A superficial biophysical assessment of A:101-121 and O-B:210-228 showed them to have dissimilar solution conformation characteristics. Although O-A:101-121 failed to strongly interact with protein components of the human epithelial intestinal cell line T84, O-B:210-228 selectively associated with occludin, claudin-one and the junctional adhesion molecule (JAM)-A. Further, the presence of O-B:210-228, but not O-A:101-121, impeded the recovery of functional TJ structures. A scrambled peptide sequences of O-B:210-228 failed to influence TJ assembly. These studies demonstrate distinct properties for these two extracellular segments of the occludin protein and provide an improved understanding of how specific domains of occludin may interact with proteins present at TJ structures.

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Figures

Figure 1.

Figure 1.

Properties of occludin synthetic peptides. (A) Cartoon of human occludin demonstrating sequence locations emulated by O-A:101–121 and O-B:210–228 peptides (detailed in Table 1). (B) Homologous peptide association studies performed with 20 μM bait peptide incubated in HBSS with Ca2+ and Mg2+ (HBSS+) in the presence or absence of 200 μM corresponding standard peptide. (C) Circular dichroism (CD) spectra of 0.2 mg standard peptide dissolved in H2O, sodium dodecylsulfate (SDS) micelles, or 92% trifluoroethanol (TFE).

Figure 2.

Figure 2.

Association of occludin bait peptides with cell proteins. Divalent cation depletion was used to dissemble tight junction (TJ) structures of polarized, confluent T84 cell monolayers. Bait peptide (200 μM O-A:101–121* or O-B:210–228*) was added at the time of Ca2+ repletion (T = 0) and monolayers were evaluated 0, 6, or 24 h later. After being washed free of unbound peptide monolayers were lysed. (A) After cell lysis and separation by SDS-PAGE, complete bait peptide labeling was determined by avidin labeling. (B) Monomeric avidin-Sepharose column-captured material was assessed by Western blot analysis. (C) Avidin-captured material was immunoprecipitated (IP) with an antibody recognizing human occludin, separated by SDS-PAGE, and Western-blotted an antibody to occludin. (D) Western blot analysis of IP occludin labeled by O-B:210–228* using antibodies that specifically recognize phospho-serine, phospho-threonine, and phosphotyrosine.

Figure 3.

Figure 3.

Localization of occludin peptides. Divalent cation depletion was used to dissemble tight junction (TJ) structures of polarized, confluent T84 cell monolayers. Bait peptide (200 μM O-A:101–121* or O-B: 210–228*) or media used for peptide addition (CTRL) was added at the time of Ca2+ repletion. After a 6- or 24-h incubation, monolayers were washed free of unbound peptide, fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100, and prepared for fluorescence microscopy. Distribution of bound bait peptides was determined by staining with FITC-conjugated streptavidin (green), whereas Alexa 568-phalloidin was used to highlight the F-actin architecture (red). Scale bar, 10 μm.

Figure 4.

Figure 4.

Effects of bait peptides on TJ architecture. Confluent T84 monolayers were subjected to a Ca2+ switch protocol with 200 μM O-A:101–121*, O-B:210–228*, or control media being added at the time of Ca2+ repletion. Immediately after (T = 0) or after 6- or 24-h incubation, monolayers were washed free of unbound peptide, fixed with ethanol or 3.7% paraformaldehyde, and prepared for fluorescence microscopy. Localization of claudin-1, occludin, JAM-A, and ZO-1 was determined by labeling with protein-specific antibodies that could be recognized by a fluorescently labeled secondary antibody (red). Distribution of bound bait peptides was determined by staining with FITC-conjugated streptavidin (green), Alexa 568-phalloidin as used to highlight the F-actin architecture (red). Scale bar, 10 μm.

Figure 5.

Figure 5.

Effects of peptides on TJ function. Divalent cation depletion was used to dissemble tight junction (TJ) structures of polarized, confluent T84 cell monolayers. (A) O-B:210–228 was added to final concentrations from 25 to 400 μM at the time of Ca2+ repletion. Addition of media used for peptide additions was used as a control (CTRL). Transepithelial electrical resistance (TER) measurements were performed over the next 24 h using “chopstick” electrodes. (B) After divalent cation depletion, 200 μM O-A:101–121, O-B:210–228, control (scrambled O-B:210–228 sequence) peptides, or control media was added to T84 monolayers at the time of Ca2+ repletion. TER measurements were made over the next 24 h using chopstick electrodes. (C) After addition of 200 μM O-A:101–121, O-B:210–228, or control media at the time of Ca2+ repletion, paracellular permeability was determined as a rate of 3-kDa fluorescent dextran transport at T = 0, 6, and 24 h.

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