Assessing the transport routes of plant proteins through the small intestinal epithelium: Utilization of novel covalently linked photo-stable labels
Liang, Outi (2025)
2025
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2025-05-22
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202505225961
https://urn.fi/URN:NBN:fi:tuni-202505225961
Tiivistelmä
In celiac disease (CD), the presence of gluten from grains causes chronic immune-mediated enteropathy affecting the small intestinal mucosa. High proline content in gluten peptides makes them partially resistant to proteolytic digestive enzymes, resulting in long gluten peptides in the gastrointestinal (GI) tract. These long gluten peptides activate unwanted immune responses in CD patients.
It has been suggested that there could be receptors for gluten peptides on the cell membranes, but none have been found. Barone et al. have done research concerning the endocytosis and transcytosis of gluten peptides and have discovered that partially digested gluten peptides enter the cells via endocytosis.
In this master’s thesis, the aim is to assess the transport routes of plant protein peptides entering the small intestinal epithelium by using new, bright, and durable labels, ATTO 565 and Qdot 655, conjugated with 6 different types of plant protein peptides. The immortalized intestinal cells, Caco-2, and induced pluripotent stem cells (iPSCs) differentiated to simple intestinal epithelial cells (SIECs), are exposed to covalently conjugated peptides. Finally, the cells are imaged either after fixing at different time points, or with time-lapse imaging to assess the transport route and locations of plant protein peptides within the epithelial cells.
The ATTO and Qdot probes have provided high stability and visibility as fluorescent labels. Due to their smaller size, the ATTO-conjugated plant proteins have proven to be a robust tool for assessing plant protein uptake by enterocytes. For now, it is found that the transcellular route dominates over the paracellular route. Plant protein peptides were detected inside cells at the 30´ time point across all peptide types, with increasing amounts observed at 60´. These results are in line with previous findings that support transcellular uptake via endocytosis. While consistent with a transcytotic route, the exact endocytic pathway was not determined. With further investigation, the exact uptake mechanism can hopefully be found.
It has been suggested that there could be receptors for gluten peptides on the cell membranes, but none have been found. Barone et al. have done research concerning the endocytosis and transcytosis of gluten peptides and have discovered that partially digested gluten peptides enter the cells via endocytosis.
In this master’s thesis, the aim is to assess the transport routes of plant protein peptides entering the small intestinal epithelium by using new, bright, and durable labels, ATTO 565 and Qdot 655, conjugated with 6 different types of plant protein peptides. The immortalized intestinal cells, Caco-2, and induced pluripotent stem cells (iPSCs) differentiated to simple intestinal epithelial cells (SIECs), are exposed to covalently conjugated peptides. Finally, the cells are imaged either after fixing at different time points, or with time-lapse imaging to assess the transport route and locations of plant protein peptides within the epithelial cells.
The ATTO and Qdot probes have provided high stability and visibility as fluorescent labels. Due to their smaller size, the ATTO-conjugated plant proteins have proven to be a robust tool for assessing plant protein uptake by enterocytes. For now, it is found that the transcellular route dominates over the paracellular route. Plant protein peptides were detected inside cells at the 30´ time point across all peptide types, with increasing amounts observed at 60´. These results are in line with previous findings that support transcellular uptake via endocytosis. While consistent with a transcytotic route, the exact endocytic pathway was not determined. With further investigation, the exact uptake mechanism can hopefully be found.