Identification of blottin: a novel gastric trefoil factor family-2 binding protein.
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.
Otto
William R
WR
Histopathology Unit, London Research Institute, Cancer Research UK, London, UK. bill.otto@cancer.org.uk
Patel
Ketan
K
McKinnell
Iain
I
Evans
Marissa D
MD
Lee
Chung-Yin
CY
Frith
David
D
Hanrahan
Sarah
S
Blight
Kenneth
K
Blin
Nikolaus
N
Kayademir
Tuncay
T
Poulsom
Richard
R
Jeffery
Rosemary
R
Hunt
Toby
T
Wright
Nicholas A
NA
McGregor
Fiona
F
Oien
Karin A
KA
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005753:Gastric Mucosa / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D036361:Peptide Hormones; D010455:Peptides / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013270:Stomach / Q000648:ultrastructure; D000071164:Trefoil Factor-2
D002352:Carrier Proteins; C477000:GKN1 protein, human; C513969:Gkn2 protein, mouse; D009124:Muscle Proteins; D036361:Peptide Hormones; D010455:Peptides; D011993:Recombinant Fusion Proteins; C000604007:TFF2 protein, human; C000604008:Tff2 protein, rat; D000071164:Trefoil Factor-2; D004720:Endonucleases; D000469:Alkaline Phosphatase