The Recognition Unit of FIBCD1 Organizes into a Noncovalently Linked Tetrameric Structure and Uses a Hydrophobic Funnel (S1) for Acetyl Group Recognition

Abstract

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.

Chitin enhances serum IgE in Aspergillus fumigatus induced allergy in mice

“…Both chitin and the partially de-acetylated version of chitin named chitosan can be phagocytised by myeloid derived cells, but the receptors responsible for this function is yet to be clearly defined (Bueter et al, 2013;Thomsen et al, 2010). Several receptors have been identified which can binds to chitin or chitin-oligosaccharides, including FIBCD1, a homotetrameric membrane protein expressed by lung and gut epithelial cells (Schlosser et al, 2009;Thomsen et al, 2010), ReGIII␥, a soluble C-type lectin (Cash et al, 2006), NKR-P1, an activating receptor on rat NK cells (Semenuk et al, 2001), and galactin-3 (Seetharaman et al, 1998). Other receptors, including toll-like receptors, the mannose receptor and dectin-1, have been suggested to modulate chitin mediated immune responses without a demonstration of direct binding to chitin (Alvarez, 2014;Lee et al, 2011).…”

Fibrinogen-Related Proteins in Tissue Repair: How a Unique Domain with a Common Structure Controls Diverse Aspects of Wound Healing

“…48 The FBG domain of FIBCD-1 forms a tetrameric structure through noncovalent interactions of adjacent molecules within the cell membrane and this form binds specifically to chitin (an N-acetyl homopolymer) through residues in the P-subdomain (Table 2), as well as interacting with GlcNAc, ManNAc, and acetylated amino acids. 48,49 After ligand binding, FIBCD-1 can endocytose acetylated compounds 48 and potential phosphorylated sites in the cytoplasmic region of this protein infer its role as a signaling molecule; however, still little is known about the events downstream of endocytosis.…”

Identification and Characterization of a Chitin-binding Protein Purified from Coelomic Fluid of the Lugworm Arenicola marina Defining a Novel Protein Sequence Family

“…Because GlcNAc-TSK was used in the initial purification, it is very likely that the initial purification from coelomic fluid only included AML-1b and excluded AML-1a. Chitin is a linear homopolymer of ␤-1,4-linked N-GlcNAc and is an important structural component in the cell wall of most fungi and in the eggshell of parasitic nematodes (43). The binding between AML-1b and chitin was calcium-independent but could be inhibited completely by GlcNAc and N-acetylated alanine but not with glucosamine or alanine (Fig.…”