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Identification of blottin: a novel gastric trefoil factor family-2 binding protein
William R Otto1, Ketan Patel, Iain McKinnell
1Histopathology Unit, London Research Institute, Cancer Research UK, London, UK. bill.otto@cancer.org.uk
Abstract:
The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.
Insights
Researchers identified a new protein, blottin, that binds to TFF2 peptides. This protein, found in the gastric epithelium, may play a role in gastrointestinal mucosal protection and cell proliferation.
Area of Science:
- Gastroenterology
- Molecular Biology
- Proteomics
Background:
- Trefoil factor family (TFF) peptides are crucial for gastrointestinal mucosal protection and repair.
- The precise mechanisms and receptors involved in TFF peptide function are not fully understood.
- Identifying TFF-binding proteins is essential for elucidating their roles in gastrointestinal health.
Purpose of the Study:
- To identify and characterize proteins that bind to TFF2.
- To investigate the potential role of TFF2-binding proteins in gastrointestinal mucosal function.
Main Methods:
- A fusion protein of mouse TFF2 with alkaline phosphatase was used to probe 2-D protein blots of mouse stomach.
- Protein identification was performed using mass spectrometry (MS).
- Characterization involved bioinformatics, rapid amplification of cDNA ends, in situ hybridization (ISH), and immunohistochemistry (IHC).
Main Results:
- A novel murine protein, named blottin, was identified as a TFF2-binding protein.
- Blottin is conserved across species (rat, human) and contains a BRICHOS domain.
- Blottin mRNA is abundant in gastric epithelium, and the protein localizes to mucus granules and Golgi stacks; it is downregulated in gastric cancers.
Conclusions:
- Blottin is a TFF2-binding protein produced by gastric epithelial cells.
- Blottin may contribute to gastrointestinal mucosal protection and modulate epithelial cell proliferation.
- Further research into blottin's function could reveal new therapeutic targets for gastrointestinal disorders.
