Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA

Abstract

Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection.

Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines

“…Exposure of Caco-2 cells to butyrate, a known transcriptional regulator of differentiation genes in many cell types, has been reported to promote the expression of the Stx receptors Gb3Cer and Gb4Cer and toxin sensitivity [ 72 ]. Interestingly, a plant-based recombinant secretory IgA antibody with binding specificity towards the Stx B-subunit, which has been developed as an edible therapeutic antibody for oral immunotherapy, was shown to neutralize the cytotoxicity of Stx1(a) towards butyrate-treated Caco-2 cells [ 96 ]. The internalization of the B-subunit of Stx1 could be significantly lowered by lipid raft disruption by cholesterol depletion.…”

Transformation strategies for stable expression of complex hetero‐multimeric proteins like secretory immunoglobulin A in plants

“…(), and the J‐chain is covalently linked into aggregates that might limit the amount of free J‐chains available to form sSIgA. A bibliographic research showed us how the described effects have been experienced previously by other authors, such as the proteolysis of IgA (Nakanishi et al ., ; Paul et al ., ; Wieland et al ., ), the simultaneous presence of different glycoforms (Juarez et al ., ; Wieland et al ., ) and the proteolysis of the SC (Ma et al ., ; Nakanishi et al ., ), the latter not observed here. These results emphasize the need to thoroughly study molecule stability and protein interactions when working with hetero‐multimeric proteins.…”

“…High SIgA levels were obtained, but three sequential crossings were needed to generate the stably transformed line containing the four different loci in homozygous condition. Using a single T‐DNA system in which all the four components required for SIgA production are clustered, significantly reduces the time to develop SIgA‐producing stable transformants as shown for Lemna (duckweed; Ariaans et al ., ) and Arabidopsis thaliana (Arabidopsis; Nakanishi et al ., ). However, the cloning process of this single T‐DNA can be challenging because of the large size of the construct.…”

Treatment Strategies for Infections With Shiga Toxin-Producing Escherichia coli

“…Another interesting concept for this strategy is the production of recombinant antibodies or even secretory antibodies specific for Stx by transgenic plants (e.g., thale grass-Arabidopsis thaliana) (Nakanishi et al, 2013(Nakanishi et al, , 2017, a concept that may be transferred to other plants, providing the possibility of an edible therapy.…”