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Identification of tubby and tubby-like protein 1 as eat-me signals by phage display.

ABSTRACT: Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed functional cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, including tubby-like protein 1 (Tulp1). This further led to the elucidation of tubby as the second eat-me signal in the same protein family. Both proteins stimulated phagocytosis of retinal pigment epithelium (RPE) cells and macrophages. Tubby-conjugated fluorescent microbeads facilitated RPE phagocytosis. Tubby and Tulp1, but not other family members, enhanced the uptake of membrane vesicles by RPE cells in synergy. Retinal membrane vesicles of Tubby mice and Tulp1(-/-) mice showed reduced activities for RPE phagocytosis, which were compensated by purified tubby and Tulp1, respectively. These data reveal a novel activity of tubby and Tulp1, and demonstrate that unbiased identification of eat-me signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.

SUBMITTER: Caberoy NB

PROVIDER: S-EPMC2789476 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Identification of tubby and tubby-like protein 1 as eat-me signals by phage display.

Caberoy Nora B NB Maiguel Dony D Kim Youngbae Y Li Wei W

Experimental cell research 20091022 2

Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed functional cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, inc ...[more]

PMID: 19837063

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Project description:Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products.Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin.We believe that several of the isolated and characterised gliadin-binding peptides described here could provide valuable tools for researchers in the field of CD by facilitating studies on localisation and uptake of various gliadin peptides in the small intestine. In future work, the potential of these peptides to detoxify gluten will be investigated.

Dataset Information

Identification of tubby and tubby-like protein 1 as eat-me signals by phage display.

Publications

Identification of tubby and tubby-like protein 1 as eat-me signals by phage display.

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