Characterization of Microfibrillar-associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-binding Protein Involved in Elastic Fiber Formation

MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization

FIBCD1 Binds Aspergillus fumigatus and Regulates Lung Epithelial Response to Cell Wall Components

Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus of clinical importance associated with development of various pulmonary diseases and allergic hypersensitivity reactions. It is protected against environmental stress by a cell wall that contains polysaccharides such as chitin. We previously demonstrated that fibrinogen C domain-containing protein 1 (FIBCD1) is a membrane-bound protein that binds chitin through a conserved S1 binding site and is expressed in intestinal epithelium and salivary glands. Here, we further localized FIBCD1 protein expression at the surface of bronchial and alveolar human lung epithelium, observed recognition of A. fumigatus cell wall with S1 site-independent recognition. We observed FIBCD1-mediated suppression of IL-8 secretion, mucin production, and transcription of genes associated with airway inflammation and homeostasis in FIBCD1-transfected lung epithelial cells. These modulations were generally enforced by stimulation with A. fumigatus cell wall polysaccharides. In parallel, we demonstrated a FIBCD1-mediated modulation of IL-8 secretion induced by TLR2,−4, and −5. Collectively, our findings support FIBCD1 as a human lung epithelial pattern recognition receptor that recognizes the complex A. fumigatus cell wall polysaccharides and modulates the lung epithelial inflammatory response by suppressing inflammatory mediators and mucins

INVESTIGATING THE MOLECULAR MECHANISMS BEHIND ON-DOWN DIRECTION-SELECTIVE GANGLION CELL CIRCUIT DEVELOPMENT

Each of the dozens of cells within the mouse retina is tuned for detecting a specific element within the visual scene. One class of retinal cell is the direction-selective ganglion cell (DSGC), which preferentially fires in response to motion in a specific direction. The molecular mechanisms that dictate DSGC directional preference remain unknown. A single-cell RNA-Seq (scRNA-Seq) experiment performed by the Kolodkin Lab compared transcriptomes of ON DSGCs that prefer upward and downward motion. Two genes enriched in upward-preferring DSGCs, Ptprk and Tbx5, are required for their normal development and function; however, the effects of genes enriched in downward-preferring DSGCs remain unexplored. The first aim of my thesis was to generate the reagents necessary for investigating ON-Down DSGCs. The lack of progress in understanding ON-Down DSGCs is due in part to a lack of mouse lines that provide genetic access to them. I achieved this by creating the Fibcd1Cre mouse line, where Cre is knocked into the locus of Fibcd1, the gene most significantly enriched in ON-Down DSGCs. These results enable the visualization and manipulation of downward-preferring DSGCs necessary for their study. The second aim of my thesis was to examine two genes enriched in ON-Down DSGCs. The first, Ptprm, encodes a receptor tyrosine phosphatase in the same subfamily as Ptprk. Loss of Ptprm, both globally and conditionally in ON-Down DSGCs, impaired the detection of upward motion. In contrast, loss of Fibcd1, which encodes a transmembrane protein that binds to acetylated compounds, had no impact on detecting visual motion; it is necessary, however, for proper ON-Down DSGC dendritic development. These results provide the first insight into the molecular mechanisms responsible for ON-Down DSGC development and function. Taken together, this work provides a foundation for the study of ON-Down DSGCs and how they acquire their specific directional preference. The Fibcd1Cre line allows for visualizing these cells and performing subtype-specific manipulations. Ptprm and Fibcd1 represent the first genes identified as tied to ON-Down DSGC development and function. These results will allow for more extensive study into the molecules that control wiring of DS circuitry

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

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 Ca2+ 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 calcium-binding 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