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Proteomic analysis of proteins surrounding occludin and claudin-4 reveals their proximity to signaling and trafficking networks.

Fredriksson K, Van Itallie CM, Aponte A, Gucek M, Tietgens AJ, Anderson JM - PLoS ONE (2015)

Bottom Line: When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins.However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex.Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tight Junction Structure and Function, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.

ABSTRACT
Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.

No MeSH data available.


Coomassie-stained SDS-PAGE gels reveal that streptavidin-purified biotinylated proteins from different transgenes show differing protein patterns.A. Shown are proteins purified from cells expressing biotin ligase alone (BL), biotin ligase fused to the N terminus of Ocln (BL-Ocln). B. Proteins purified from the N- and C- terminus of Ocln (BL-Ocln and Ocln-BL) with added biotin. C. Shown are proteins purified from cells expressing biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4), with or without added biotin. The positions of the transgenes are marked with arrowheads. Triplicate samples gave very similar protein patterns.
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pone.0117074.g003: Coomassie-stained SDS-PAGE gels reveal that streptavidin-purified biotinylated proteins from different transgenes show differing protein patterns.A. Shown are proteins purified from cells expressing biotin ligase alone (BL), biotin ligase fused to the N terminus of Ocln (BL-Ocln). B. Proteins purified from the N- and C- terminus of Ocln (BL-Ocln and Ocln-BL) with added biotin. C. Shown are proteins purified from cells expressing biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4), with or without added biotin. The positions of the transgenes are marked with arrowheads. Triplicate samples gave very similar protein patterns.

Mentions: To determine whether there is specificity to the proteins labeled by the biotin ligase fusion proteins and purified on streptavidin resin, we first compared the pattern of purified proteins on SDS-PAGE gels from cells expressing biotin ligase alone (Fig. 3A, left panels) with proteins from cells expressing BL-Ocln (Fig. 3A, right panels). This analysis demonstrated that the pattern of recovered proteins was dependent on induction of the specific biotin ligase fusion protein and on the addition of biotin to the cell cultures. Protein bands from un-induced cells, and bands from cells in the absence of added biotin, likely reflect the presence of endogenously biotinylated proteins, including carboxylases [48]. The pattern of these bands of endogenously biotinylated proteins in all controls (un-induced cells without added biotin, induced cells without added biotin, and un-induced cells with added biotin) appear very similar (Fig. 3A). In contrast, the pattern of Coomassie-stained proteins from cells induced to express biotin ligase fusion proteins and incubated with exogenous biotin reveals novel protein patterns that varies among the different fusion proteins (for example, compare Fig. 3A, lanes 4 and 8). The marked reduction of many of the endogenously biotinylated protein bands in samples from cells with induced biotin ligase fusion-proteins, and added biotin, maybe due to competition for binding to the streptavidin resin.


Proteomic analysis of proteins surrounding occludin and claudin-4 reveals their proximity to signaling and trafficking networks.

Fredriksson K, Van Itallie CM, Aponte A, Gucek M, Tietgens AJ, Anderson JM - PLoS ONE (2015)

Coomassie-stained SDS-PAGE gels reveal that streptavidin-purified biotinylated proteins from different transgenes show differing protein patterns.A. Shown are proteins purified from cells expressing biotin ligase alone (BL), biotin ligase fused to the N terminus of Ocln (BL-Ocln). B. Proteins purified from the N- and C- terminus of Ocln (BL-Ocln and Ocln-BL) with added biotin. C. Shown are proteins purified from cells expressing biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4), with or without added biotin. The positions of the transgenes are marked with arrowheads. Triplicate samples gave very similar protein patterns.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366163&req=5

pone.0117074.g003: Coomassie-stained SDS-PAGE gels reveal that streptavidin-purified biotinylated proteins from different transgenes show differing protein patterns.A. Shown are proteins purified from cells expressing biotin ligase alone (BL), biotin ligase fused to the N terminus of Ocln (BL-Ocln). B. Proteins purified from the N- and C- terminus of Ocln (BL-Ocln and Ocln-BL) with added biotin. C. Shown are proteins purified from cells expressing biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4), with or without added biotin. The positions of the transgenes are marked with arrowheads. Triplicate samples gave very similar protein patterns.
Mentions: To determine whether there is specificity to the proteins labeled by the biotin ligase fusion proteins and purified on streptavidin resin, we first compared the pattern of purified proteins on SDS-PAGE gels from cells expressing biotin ligase alone (Fig. 3A, left panels) with proteins from cells expressing BL-Ocln (Fig. 3A, right panels). This analysis demonstrated that the pattern of recovered proteins was dependent on induction of the specific biotin ligase fusion protein and on the addition of biotin to the cell cultures. Protein bands from un-induced cells, and bands from cells in the absence of added biotin, likely reflect the presence of endogenously biotinylated proteins, including carboxylases [48]. The pattern of these bands of endogenously biotinylated proteins in all controls (un-induced cells without added biotin, induced cells without added biotin, and un-induced cells with added biotin) appear very similar (Fig. 3A). In contrast, the pattern of Coomassie-stained proteins from cells induced to express biotin ligase fusion proteins and incubated with exogenous biotin reveals novel protein patterns that varies among the different fusion proteins (for example, compare Fig. 3A, lanes 4 and 8). The marked reduction of many of the endogenously biotinylated protein bands in samples from cells with induced biotin ligase fusion-proteins, and added biotin, maybe due to competition for binding to the streptavidin resin.

Bottom Line: When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins.However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex.Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tight Junction Structure and Function, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.

ABSTRACT
Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.

No MeSH data available.