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Paracellular Transport of Soybean β‐Conglycinin using a Caco‐2/HT29‐MTX Co‐Culture
Author(s) -
Wallace Frances Lynn,
Mahler Gretchen J.,
TanWilson Anna,
Wilson Karl A.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.794.10
Subject(s) - tight junction , paracellular transport , caco 2 , occludin , protein subunit , chemistry , storage protein , microbiology and biotechnology , intestinal epithelium , polyclonal antibodies , barrier function , biochemistry , epithelium , biophysics , cell , permeability (electromagnetism) , biology , antibody , immunology , membrane , genetics , gene
Soybeans are one of the eight most prevalent food allergens and the β‐conglycinins have been determined to be one of the allergenic proteins. β‐conglycinins are homo‐ or hetero‐ trimers of evolutionarily related α, α’ and β subunits. The allergenic response to seed storage proteins is thought to be due in part to the passage of undigested proteins passing through the tight junction gaps in the gut epithelium, where they are accessible to gut associated lymphoid tissue. The goal of this study is to compare the passage of homo‐ and hetero‐trimers of β‐conglycinin through a gastrointestinal epithelium model to determine whether the cell barrier will have an altered permeability to β‐conglycinins of different subunit compositions. The native β‐conglycinins were purified from soybean seeds and further separated into trimers of only α, α’, and β subunits. A Transwell plate was constructed using Caco‐2 and HT29‐MTX cells in a ratio of 3:1. The integrity of the tight junctions of the cell monolayer was gauged by measuring transepithelial resistance (TER) and occludin expression. To measure protein passage through the cell monolayer, the proteins were applied to the apical chamber and the medium collected after a period of 2 h from both the apical and basolateral chambers. The amounts of protein in both chambers were quantified using rabbit polyclonal antibodies specific to the soybean β‐conglycinins. The passage of β‐conglycinins of different subunit compositions are compared as a function of TER. The permeability of the cell monolayer can be increased by different means, such as varying the time period between cell seeding and transport experiments and chemical inhibitors. When the cell monolayer showed a TER of 250–300 ohms/cm 2 , there was no detectable protein transport.