Mechanisms of Expression and Internalisation of FIBCD1; a novel Pattern Recognition Receptor in the Gut Mucosa.

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Background Fibrinogen C domain-containing protein 1 (FIBCD1) is a type II transmembrane protein lo¬cated on the luminal side of epithelial cells in the intestinal mucosa. FIBCD1 binds specifically to acetylated compounds through the C-terminal fibrinogen-re¬lated domain (FReD). The FReD domain is a carbohydrate recognition domain also expressed by the ficolins, which are pattern recognition molecules that activate the complement system via the lectin pathway. Chitin is a highly ace¬tylated homopolymer of β-1,4-N-acetyl-glucosamine carbohydrate found abundantly in nature in organisms such as fungi and parasitic nematodes. In vitro we have shown that FIBCD1 binds chitin and internalises the soluble model ligand acetylated BSA and directs it for endosomal degradation. In vivo we have shown that FIBCD1 has a defined role in gut homeostasis, but to date, the molecular mechanisms behind remain unknown.
Aim In this project we aim to characterise the functional aspects of the binding between FIBCD1 and chitin. We have constructed FIBCD1-expressing epithelial cell lines in which we seek to identify the intracellular signalling upon binding to chitin or to a model ligand. In vivo studies will include the fibcd1-knockout mouse generated by our lab. The mechanisms of internalisation of acetylated structures mediated by FIBCD1 will be investigated by performing a range of strategic point mutations in the cytoplasmic tail of FIBCD1.
Results The colon epithelial cell line HCT-116 has been stably transfected with full length FIBCD1 in order to use for stimulatory studies and the cell line HEK293 has been stably transfected with FIBCD1 containing point mutations in the cytoplasmic tail in use for internalisation studies.
Preliminary results show that FIBCD1 internalisation of monoclonal antibody or acetylated BSA can be inhibited by sucrose and chlorpromazine and not by filipin and nystatin. This indicates that FIBCD1 is internalised via clathrin-coated pits and not via caveolae. Deletion of the proximal 25 amino acids of the N-terminal results in absence of expression. If only the proximal 20 amino acids are deleted, expression and function of FIBCD1 is fully preserved. This observation indicates that the amino acids 20-25 comprise a putative motif necessary for expression and/or translocation to the cell membrane.
Conclusion We have identified FIBCD1 as the first membrane bound FReD molecule in mam¬mals and the first pattern recognition receptor that binds chitin and directs acetylated structures for de¬gradation in the endosome via clathrin-mediated endocytosis. The localisation of FIBCD1 in the intestinal mucosal epithelia points towards a functional role in innate immunity and/or gut homeostasis.
Original languageEnglish
Publication date2012
Number of pages1
Publication statusPublished - 2012

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