Identification of blottin: A novel gastric trefoil factor family-2 binding protein

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.

Cytokine Regulation of the Trefoil Factor Family Binding Protein GKN2 (GDDR/TFIZ1/blottin) in Human Gastrointestinal Epithelial Cells

“…Electron microscopy showed that mouse blottin (Gkn2) is being secreted with mucin granules and co-localized with TFF1. Interestingly TFF2 is found deeper in the gastric glands [17]. Blottin's human homologue TFIZ1/ GDDR, is down-regulated in gastric cancer, as suggested by original reports [18,19].…”

Evolution of the human gastrokine locus and confounding factors regarding the pseudogenicity ofGKN3

“…GKN2 differs from GKN1 in part by its ability to covalently bind TFF1 via a cysteine bond residue C38 and thus inhibit the growth of cancer cells (7,19,52). GKN2 has been reported to noncovalently interact with TFF2 in mouse stomach tissue, but this does not appear to occur in human antral tissue (19,32,33). Like GKN1 and TFF1, GKN2 is expressed in surface pit cells in both the corpus and antrum and thought to be secreted (19,32,52).…”

“…GKN2 has been reported to noncovalently interact with TFF2 in mouse stomach tissue, but this does not appear to occur in human antral tissue (19,32,33). Like GKN1 and TFF1, GKN2 is expressed in surface pit cells in both the corpus and antrum and thought to be secreted (19,32,52). In contrast, TFF2 is expressed in the mucous neck region of corporal units and in the base of antral units, suggesting any interactions with GKN2 would occur in the gastric lumen (19,29).…”

Secreted Trefoil Factor 2 Activates the CXCR4 Receptor in Epithelial and Lymphocytic Cancer Cell Lines

“…Two TFF2-binding proteins that have been characterized include a 140-kDa protein, the ␤ subunit of the fibronectin receptor, and a 224-kDa protein called muclin (15). Another TFF2-binding protein was isolated by probing two-dimensional blots of mouse stomach with a murine TFF2 fusion protein, leading to the identification of the gastric foveolar protein blottin, a murine homolog of the human peptide TFIZ1 (16). Although these three proteins have now been well characterized, none of them has been shown to mediate responses to TFF2, and no activated signaling cascades have been shown.…”