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...

Lipopolysaccharides (LPS) of Gram-negative bacteria are important mediators of bacterial virulence that can elicit potent endotoxic effects. Surfactant protein D (SP-D) shows specific interactions with LPS, both in vitro and in vivo. These interactions involve binding of the carbohydrate recognition domain (CRD) to LPS oligosaccharides (OS); however, little is known about the mechanisms of LPS recognition. Recombinant neck+CRDs (NCRDs) provide an opportunity to directly correlate binding interactions with a crystallographic analysis of the binding mechanism. In these studies, we examined the interactions of wild-type and mutant trimeric NCRDs with rough LPS (R-LPS). Although rat NCRDs bound more efficiently than human NCRDs to Escherichia coli J-5 LPS, both proteins exhibited efficient binding to solid-phase Rd2-LPS and to Rd2-LPS aggregates presented in the solution phase. Involvement of residues flanking calcium at the sugar binding site was demonstrated by reciprocal exchange of lysine and arginine at position 343 of rat and human CRDs. The lectin activity of hNCRDs was inhibited by specific heptoses, including l-glycero-α-d-manno-heptose (l,d-heptose), but not by 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo). Crystallographic analysis of the hNCRD demonstrated a novel binding orientation for l,d-heptose, involving the hydroxyl groups of the side chain. Similar binding was observed for a synthetic α1→3-linked heptose disaccharide corresponding to heptoses I and II of the inner core region in many LPS. 7-O-Carbamoyl-l,d-heptose and d-glycero-α-d-manno-heptose were bound via ring hydroxyl groups. Interactions with the side chain of inner core heptoses provide a potential mechanism for the recognition of diverse types of LPS by SP-D.

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