Structural and Functional Studies on the Interaction of Adenovirus Fiber Knobs and Desmoglein 2

Abstract

f Human adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14, as well as a recently emerged strain of Ad14 (Ad14p1), use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. Unlike Ad interaction with CAR and CD46, structural details for Ad binding to DSG2 are still elusive. Using an approach based on Escherichia coli expression libraries of random Ad3 and Ad14p1 fiber knob mutants, we identified amino acid residues that, when mutated individually, ablated or reduced Ad knob binding to DSG2. These residues formed three clusters inside one groove at the extreme distal end of the fiber knob. The Ad3 fiber knob mutant library was also used to identify variants with increased affinity to DSG2. We found a number of mutations within or near the EF loop of the Ad3 knob that resulted in affinities to DSG2 that were several orders of magnitude higher than those to the wild-type Ad3 knob. Crystal structure analysis of one of the mutants showed that the introduced mutations make the EF loop more flexible, which might facilitate the interaction with DSG2. We recently identified DSG2 as the main receptor for a group of species B adenoviruses, including adenovirus serotype 3 (Ad3), a serotype which is widely distributed in the human population (1). We found that the DSG2-interacting domain(s) within Ad3 is formed by several fiber knobs (2). This specific mode of Ad3 fiber knob-DSG2 interaction provides a high avidity and is functionally relevant for opening of epithelial junctions (1, 2). The latter involves clustering of DSG2 and activation of pathways that are reminiscent of an epithelial-to-mesenchymal transition, including the phosphorylation of mitogen-activated protein kinase (MAPK) and the downregulation of junction protein expression (1,3,4). The ability to open epithelial junctions appears to be important for Ad3 penetration into and spread within airway epithelial cells (1, 2, 4). In a recent study, we attempted to find the minimal moiety within the Ad3 capsid that confers efficient binding to DSG2 (2). We generated a small recombinant protein which contains the Ad3 fiber knob and a domain that allows for the self-dimerization of trimeric Ad3 fiber knobs (JO-1) (2). JO-1 can be readily produced in Escherichia coli and purified by affinity chromatography. In polarized epithelial cell cultures, JO-1 triggered the opening of intercellular junctions, while intravenous injection of JO-1 into mice with epithelial tumors allowed for better penetration of anti-cancer drugs (3, 5).The first goal of the present study was to further delineate structural features of the Ad3 fiber knob-DSG2 interaction. This included identifying the amino acid residues within the Ad3 fiber knob that are involved in binding to DSG2 and creating JO-1 mutants with reduced and ablated binding to DSG2. The second goal of this study, which has translational relevance, was to further improve JO-1 by enhancing its affinity to DSG2, thereby increasing its therapeutic effect. This was done by identifying mutants with increased b...

Intracellular Signaling and Desmoglein 2 Shedding Triggered by Human Adenoviruses Ad3, Ad14, and Ad14P1

“…Another DSG2-interacting species B Ad is Ad14 and its recently emerged strain Ad14P1 (33). Like Ad3, Ad14 and Ad14P1 generate PtDd during infection (30).…”

“…This was achieved by linking the coding sequence of the Ad3 fiber knob to a K-coil domain. A protein that contained the K-coil domain and Ad3 fiber knob was called JO1 (33). JO1 can be purified by affinity chromatography.…”

Cell–Cell Junctions Organize Structural and Signaling Networks

“…Once bound, MAP kinases activate the extracellular matrix metalloproteinase ADAM17, which cleaves the extracellular domain of Dsg2 (Fig. 5; Wang et al 2013, 2015); this process has been used as an ectopic procedure to increase the penetration of therapeutic drugs into the skin (Yumul et. al 2016).…”

Galectin-3 Regulates Desmoglein-2 and Intestinal Epithelial Intercellular Adhesion

“…Intercellular junctions are actively remodeled during epithelial proliferation, migration, and differentiation. Intercellular junction proteins regulate signaling events to control these biological processes (1)(2)(3)(4)(5)(6). Thus, altered function of intercellular junction proteins contributes not only to the compromised epithelial barrier but also to changes in epithelial homeostasis observed in disease states associated with mucosal inflammation and neoplasia (1,(7)(8)(9)(10)(11).…”