Chitin is a highly acetylated compound and the second most abundant biopolymer in the world next to cellulose. Vertebrates are exposed to chitin both through food ingestion and when infected with parasites, and fungi and chitin modulate the immune response in different directions. We have identified a novel homotetrameric 55-kDa type II transmembrane protein encoded by the FIBCD1 gene and highly expressed in the gastrointestinal tract. The ectodomain of FIBCD1 is characterized by a coiled-coil region, a polycationic region and C-terminal fibrinogen-related domain that by disulfide linkage assembles the protein into tetramers. Functional analysis showed a high-affinity and calcium-dependent binding of acetylated components to the fibrinogen domain, and a function in endocytosis was demonstrated. Screening for ligands revealed that the FIBCD1 is a high-affinity receptor for chitin and chitin fragments. FIBCD1 may play an important role in controlling the exposure of intestine to chitin and chitin fragments, which is of great relevance for the immune defense against parasites and fungi and for immune response modulation.

Pulmonary surfactant protein A (SP‐A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro‐organisms, and mediates microbial lysis and clearance. SP‐A also modulates multiple immune‐related functions including cytokine production and chemotaxis for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril‐associated protein 4 (MFAP4) and SP‐A. MFAP4 is a collagen‐binding molecule containing a C‐terminal fibrinogen‐like domain and a N‐terminal located integrin‐binding motif. We produced recombinant MFAP4 with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium‐dependent binding between MFAP4 and human SP‐A1 and SP‐A2. No binding was seen to recombinant SP‐A composed of the neck region and carbohydrate recognition domain of SP‐A indicating that the interaction between MFAP4 and SP‐A is mediated via the collagen domain of SP‐A. Monoclonal antibodies directed against MFAP4 and SP‐A were used for immunohistochemical analysis, which demonstrates that the two molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP‐A via the collagen region in vitro, and that MFAP4 and SP‐A colocates in different lung compartments indicating that the interaction may be operative in vivo.

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We have recently identified FIBCD1 (Fibrinogen C domain containing 1) as a type II transmembrane endocytic receptor located primarily in the intestinal brush border. The ectodomain of FIBCD1 comprises a coiled coil, a polycationic region, and a C-terminal FReD (fibrinogen-related domain) that assembles into disulfide-linked homotetramers. The FIBCD1-FReD binds Ca 2؉ dependently to acetylated structures like chitin, N-acetylated carbohydrates, and amino acids. FReDs are present in diverse innate immune pattern recognition proteins including the ficolins and horseshoe crab TL5A. Here, we use chemical cross-linking, combined with analytical ultracentrifugation and electron microscopy of the negatively stained recombinant FIBCD1-FReD to show that it assembles into noncovalent tetramers in the absence of the coiled coil. We use surface plasmon resonance, carbohydrate binding, and pulldown assays combined with site-directed mutagenesis to define the binding site involved in the interaction of FIBCD1 with acetylated structures. We show that mutations of central residues (A432V and H415G) in the hydrophobic funnel (S1) abolish the binding of FIBCD1 to acetylated bovine serum albumin and chitin. The double mutations (D393N/D395A) at the putative calciumbinding site reduce the ability of FIBCD1 to bind ligands. We conclude that the FReDs of FIBCD1 forms noncovalent tetramers and that the acetyl-binding site of FReDs of FIBCD1 is homologous to that of tachylectin 5A and M-ficolin but not to the FReD of L-ficolin. We suggest that the spatial organization of the FIBCD1-FReDs determine the molecular pattern recognition specificity and subsequent biological functions.