Recognition of Mannosylated Ligands and Influenza A Virus by Human Surfactant Protein D: Contributions of an Extended Site and Residue 343^,

Abstract

Surfactant protein D (SP-D) plays important roles in antiviral host defense. Although SP-D shows a preference for glucose/maltose, the protein also recognizes D-mannose and a variety of mannoserich microbial ligands. This latter preference prompted an examination of the mechanisms of mannose recognition, particularly as they relate to high-mannose viral glycans. Trimeric neck +carbohydrate recognition domains from human SP-D (hNCRD) preferred alpha1-2 linked dimannose (DM) over the branched trimannose (TM) core, alpha1-3 or alpha1-6 DM, or D-mannose. Previous studies have shown residues flanking the carbohydrate binding site can fine-tune ligand recognition. A mutant with valine at 343 (R343V) showed enhanced binding to mannan relative to wild-type and R343A. No alteration in affinity was observed for D-mannose or for alpha1-3 or alpha1-6 linked DM; however, substantially increased affinity was observed for alpha1-2DM. Both proteins showed efficient recognition of linear and branched sub-domains of high-mannose glycans on carbohydrate microarrays, and R343V showed increased binding to a subset of the oligosaccharides. Crystallographic analysis of an R343V complex with 1,2-DM showed a novel mode of binding. The disaccharide is bound to calcium by the reducing sugar ring, and a stabilizing H-bond is formed between the 2-OH of the non-reducing sugar ring and Arg349. Although hNCRDs show negligible binding to influenza A virus (IAV), R343V showed markedly enhanced viral neutralizing activity. Hydrophobic substitutions for Arg343 selectively blocked binding of a monoclonal antibody (Hyb 246-05) that inhibits IAV binding activity. Our findings demonstrate an extended ligand binding site for mannosylated ligands and the significant contribution of the 343 side chain to specific recognition of multivalent microbial ligands, including high-mannose viral glycans.SP-D is an effector of antimicrobial host defense in the lung and at extrapulmonary sites of expression (1)(2)(3)(4)(5). Animals deficient in SP-D are more susceptible to pulmonary challenge with † This publication was made possible by Grant Number HL-44015 and HL-29594 (EC) NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 21. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptPseudomonas aeruginosa (6), and intranasal administration of recombinant SP-D can protect wild-type mice from lethal challenge with Aspergillus fumigatus (7). In addition, SP-D appears to play particularly important roles in the neutralization and clearance of viruses. Animals deficient in SP-D also show delayed clearance and heightened inflammatory responses to strains of influenza A virus (IAV) and respiratory syncytial virus (RSV) (8,9). Clearance is selectively impaired for IAV strains reactive with SP-D in vitro (8) and can be rescued with recombinant SP-D or by transgenic overexpression of trimeric subunits of 10). Notably, specific polymorphisms in the human gene have been associated with enhanced inflamm...

Mutations flanking the carbohydrate binding site of surfactant protein D confer antiviral activity for pandemic influenza A viruses

“…&Maltose significantly reduced the inhibitory activity of D325AϩR343V (P Ͻ 0.05; n ϭ 4). certain glycans to the lectin site and to accommodate alternative binding orientations, there is a clear requirement for a hydrophobic residue at this position (5). Our present studies strongly support the hypothesis that the Asp325 side chain normally limits access of viral glycans to the lectin site of wild-type SP-D. Substitutions with alanine or serine showed modestly enhanced interactions with highly glycosylated seasonal strains of IAV, whereas D325V showed no enhancement.…”

“…We and others have studied the activity of recombinant versions of SP-D composed of only the neck and carbohydrate recognition domains (NCRDs) of the molecule, without the extended collagen domain or NH 2 -terminus (3,5,7,16,20,28). The advantages of such NCRDs include greater ability to directly compare activity between different mutants (i.e., by eliminating the complicating effects of variable higherorder multimerization conferred by the collagen and NH 2 -terminal domains), the ability to crystallize NCRDs and perform structure-function analysis, and greater ease of production.…”

Crystal Structure of a Complex of Surfactant Protein D (SP-D) and Haemophilus influenzae Lipopolysaccharide Reveals Shielding of Core Structures in SP-D-Resistant Strains

“…3b). Thus, recognition of the Eagan 4A inner core involves not only the primary calcium ion but also both binding site flanking residues (20)(21)(22)(23)(24). This concerted binding suggests high affinity for the HepI-Kdo disaccharide, consistent with the binding studies where Eagan 4A competes strongly with the high-affinity SP-D ligand maltose.…”

“…rfhSP-D retains significant biological activity in vitro and in vivo (17,18), exhibiting therapeutic activity in murine models of pulmonary hypersensitivity and infection induced by an opportunistic fungus, Aspergillus fumigatus (19). Reported structures of ligandbound complexes are restricted to simple but representative ligands (20)(21)(22)(23)(24). Recognition is achieved through CRD Cadependent binding of the terminal monosaccharide through a mannose-type equatorial hydroxyl pair or, as in the case of inositol-1-phosphate, galactose and L,D-heptose, a stereochemically equivalent pair.…”

A Unique Sugar-binding Site Mediates the Distinct Anti-influenza Activity of Pig Surfactant Protein D

“…For RhNCRD, simulations started with coordinates from RhNCRD (PDB code 3G83 (53), with the ligand removed), with or without docked Man 8 . After 65-ns simulations, the average root mean square deviations (r.m.s.d.)…”