Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2011; 286(47):40681-92. DOI: 10.1074/jbc.M111.300673
Source: PubMed


The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.

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    • "Alignment of serum collectins with SP-D has also revealed nonconservative substitutions at amino acid positions 325 and 343 which are highly exposed on opposing ridges of the carbohydrate-binding groove [140]. Recent studies have substituted these residues (alone or in combination) into the SP-D NCRD in an attempt to enhance ligand binding and anti-IAV activity [54, 116, 138, 140]. Crouch et al. recently demonstrated that a mutant of the human SP-D NCRD that contained 2 amino acid substitutions (D325A + R343V) displayed enhanced anti-IAV activity in vitro compared to mutants which expressed either substitution alone [140]. "
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