Barbara McDonald

Ligand Specificity of Human Surfactant Protein D EXPRESSION OF A MUTANT TRIMERIC COLLECTIN THAT SHOWS ENHANCED INTERACTIONS WITH INFLUENZA A VIRUS

Surfactant protein D is a pattern recognition molecule that plays diverse roles in immune regulation and anti-microbial host defense. Its interactions with known ligands are calcium-dependent and involve binding to the trimeric, C-type carbohydrate recognition domain. Surfactant protein D preferentially binds to glucose and related sugars. However, CL-43, a bovine serum lectin, which evolved through duplication of the surfactant protein D gene in ruminants, prefers mannose and mannose-rich polysaccharides. Surfactant protein D is characterized by two relatively conserved motifs at the binding face, along the edges of the shallow carbohydrate-binding groove. For CL-43, sequence alignments demonstrate a basic insertion, Arg-Ala-Lys (RAK), immediately N-terminal to the first motif. We hypothesized that this insertion contributes to the differences in saccharide selectivity and host defense function and compared the activities of recombinant trimeric neck + carbohydrate recognition domains of human surfactant protein D (NCRD) with CL-43 (RCL-43-NCRD) and selected NCRD mutants. Insertion of the CL-43 RAK sequence or a control Ala-Ala-Ala sequence (AAA) into the corresponding position in NCRD increased the efficiency of binding to mannan and changed the inhibitory potencies of competing saccharides to more closely resemble those of CL-43. In addition, RAK resembled CL-43 in its greater capacity to inhibit the infectivity of influenza A virus and to increase uptake of influenza by neutrophils.

Modification of surfactant protein D by reactive oxygen-nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo

Surfactant protein D (SP‐D) is an important effector of innate immunity. We have previously shown that SP‐D accumulates at sites of acute bacterial infection and neutrophil infiltration, a setting associated with the release of reactive species such as peroxynitrite. Incubation of native SP‐D or trimeric SP‐D lectin domains (NCRDs) with peroxynitrite resulted in nitration and nondisulfide cross‐linking. Modifications were blocked by peroxynitrite scavengers or pH inactivation of peroxynitrite, and mass spectroscopy confirmed nitration of conserved tyrosine residues within the C‐terminal neck and lectin domains. Mutant NCRDs lacking one or more of the tyrosines allowed us to demonstrate preferential nitration of Tyr314 and the formation of Tyr228‐dependent cross‐links. Although there was no effect of peroxynitrite or tyrosine mutations on lectin activity, incubation of SP‐D dodecamers or murine lavage with peroxynitrite decreased the SP‐D‐dependent aggregation of lipopolysaccharide‐coated beads, supporting our hypothesis that defective aggregation results from abnormal cross‐linking. We also observed nitration, cross‐linking of SP‐D, and a significant decrease inSP‐D‐dependent aggregating activity in the lavage of mice acutely exposed to nitrogen dioxide. Thus, modification of SP‐D by reactive oxygen‐nitrogen species could contribute to alterations in the structure and function of SP‐D at sites of inflammation in vivo.— Matalon, S., Shrestha, K., Kirk, M., Waldheuser, S., McDonald, B., Smith, K., Gao, Z., Belaaouaj, A., Crouch, E. C. Modification of surfactant protein D by reactive oxygen‐nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo. FASEB J. 23, 1415–1430 (2009)

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

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 mannose-rich 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 plus 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 alpha1-2 DM. Both proteins showed efficient recognition of linear and branched subdomains 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 nonreducing 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.

Patterns of neutrophil serine protease-dependent cleavage of surfactant protein D in inflammatory lung disease

The manuscript presents definitive studies of surfactant protein D (SP‐D) in the context of inflammatory lung fluids. The extent of SP‐D depletion in bronchoalveolar lavage fluid (BALF) of children affected with cystic fibrosis (CF) is demonstrated to correlate best with the presence of the active neutrophil serine protease (NSP) elastase. Novel C‐terminal SP‐D fragments of 27 kDa and 11 kDa were identified in patient lavage fluid in addition to the previously described N‐terminal, 35‐kDa fragment by the use of isoelectrofocusing, modified blotting conditions, and region‐specific antibodies. SP‐D cleavage sites were identified. In vitro treatment of recombinant human SP‐D dodecamers with NSPs replicated the fragmentation, but unexpectedly, the pattern of SP‐D fragments generated by NSPs was dependent on calcium concentration. Whereas the 35‐ and 11‐kDa fragments were generated when incubations were performed in low calcium (200 μM CaCl2), incubations in physiological calcium (2 mM) with higher amounts of elastase or proteinase‐3 generated C‐terminal 27, 21, and 14 kDa fragments, representing cleavage within the collagen and neck regions. Studies in which recombinant SP‐D cleavage by individual NSPs was quantitatively evaluated under low and high calcium conditions showed that the most potent NSP for cleaving SP‐D is elastase, followed by proteinase‐3, followed by cathepsin G. These relative potency findings were considered in the context of other studies that showed that active NSPs in CF BALF are in the order: elastase, followed by cathepsin G, followed by proteinase‐3. The findings support a pre‐eminent role for neutrophil elastase as the critical protease responsible for SP‐D depletion in inflammatory lung disease.

Contributions of phenylalanine 335 to ligand recognition by human surfactant protein D : Ring interactions with SP-D ligands

Surfactant protein D (SP-D) is an innate immune effector that contributes to antimicrobial host defense and immune regulation. Interactions of SP-D with microorganisms and organic antigens involve binding of glycoconjugates to the C-type lectin carbohydrate recognition domain (CRD). A trimeric fusion protein encoding the human neck+CRD bound to the aromatic glycoside p-nitrophenyl-α-d-maltoside with nearly a log-fold higher affinity than maltose, the prototypical competitor. Maltotriose, which has the same linkage pattern as the maltoside, bound with intermediate affinity. Site-directed substitution of leucine for phenylalanine 335 (Phe-335) decreased affinities for the maltoside and maltotriose without significantly altering the affinity for maltose or glucose, and substitution of tyrosine or tryptophan for leucine restored preferential binding to maltotriose and the maltoside. A mutant with alanine at this position failed to bind to mannan or maltose-substituted solid supports. Crystallographic analysis of the human neck+CRD complexed with maltotriose or p-nitrophenyl-maltoside showed stacking of the terminal glucose or nitrophenyl ring with the aromatic ring of Phe-335. Our studies indicate that Phe-335, which is evolutionarily conserved in all known SP-Ds, plays important, if not critical, roles in SP-D function.

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

Background: SP-D plays important roles in the defense against influenza A. Results: A recombinant SP-D with combinatorial mutations shows enhanced interactions with hemagglutinin-associated glycans and augmented antiviral activity in vitro and in vivo. Conclusion: Exogenous forms of recombinant SP-D can rescue mice from a lethal challenge with influenza. Significance: It may be possible to develop collectin-based interventions for influenza. 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.

Myeloperoxidase-dependent Inactivation of Surfactant Protein D in Vitro and in Vivo

Surfactant protein D (SP-D) plays diverse and important roles in innate immunity and pulmonary homeostasis. Neutrophils and myeloperoxidase (MPO) colocalized with SP-D in a murine bacterial pneumonia model of acute inflammation, suggesting that MPO-derived reactive species might alter the function of SP-D. Exposure of SP-D to the complete MPO-H2O2-halide system caused loss of SP-D-dependent aggregating activity. Hypochlorous acid (HOCl), the major oxidant generated by MPO, caused a similar loss of aggregating activity, which was accompanied by the generation of abnormal disulfide-cross-linked oligomers. A full-length SP-D mutant lacking N-terminal cysteine residues and truncation mutants lacking the N-terminal domains were resistant to the oxidant-induced alterations in disulfide bonding. Mass spectroscopy of HOCl-treated human SP-D demonstrated several modifications, but none involved key ligand binding residues. There was detectable oxidation of cysteine 15, but no HOCl-induced cysteine modifications were observed in the C-terminal lectin domain. Together, the findings localize abnormal disulfide cross-links to the N-terminal domain. MPO-deficient mice showed decreased cross-linking of SP-D and increased SP-D-dependent aggregating activity in the pneumonia model. Thus, MPO-derived oxidants can lead to modifications of SP-D structure with associated alterations in its characteristic aggregating activity.

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

We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.

Enhancement of Antiviral Activity of Collectin Trimers through Cross-Linking and Mutagenesis of the Carbohydrate Recognition Domain

Surfactant protein D (SP-D) plays important roles in innate defense against respiratory viruses [including influenza A viruses (IAVs)]. Truncated trimers composed of its neck and carbohydrate recognition domains (NCRDs) bind various ligands; however, they have minimal inhibitory activity for IAV. We have sought to find ways to increase the antiviral activity of collectin NCRDs. Cross-linking of the SP-D NCRD with nonblocking monoclonal antibodies (mAbs) markedly potentiates antiviral activity. In the present report, we demonstrate that F(ab’)2 [but not F(ab’)1] fragments of a cross-linking mAb have similar effects. Hence, cross-linking activity, but not the Fc domain of the mAb, is needed for increased antiviral activity. In contrast, the Fc domain of the mAb was important for increasing viral uptake or respiratory burst responses of human neutrophils. Our NCRD constructs contain an S protein binding site. Herein, we show that a multivalent S protein complex caused cross-linking and also increased the antiviral activity of NCRDs. NCRDs of conglutinin and CL43 had greater intrinsic antiviral activity than those of SP-D or mannose-binding lectin. Based on motifs found in these serum collectins, we have constructed mutant versions of the human SP-D NCRD that have increased antiviral activity. These mutant NCRDs also had potentiated activity after cross-linking with F(ab’)2 fragments or S protein complexes. Hence, the antiviral activity of NCRDs can be increased by 2 distinct, complementary strategies, namely cross-linking of NCRDs through various means and mutagenesis of CRD residues to increase viral binding. These findings may be relevant for antiviral therapy.

034 Myeloperoxidase mediates oxidation of surfactant protein-D abrogating its biological activities

Introduction Surfactant protein D (SP-D) is expressed in the lung and plays important roles in innate immunity including defense against inhaled pathogens. While the levels of SP-D have been shown to decrease in acutely injured lung, little is known about the mechanism(s) of its inactivation and clearance. A hall-mark of acute inflammation is the recruitment and activation of polymorphonuclear neutrophils (PMNs). Activated PMNs employ different pathways to generate toxic oxidants with deleterious effect on host tissues. In the myeloperoxidase (MPO) pathway, MPO employs hydrogen peroxide (H2O2) and chloride to generate HOC1, a highly reactive and toxic oxidant. These observations prompted us to hypothesize that MPO-derived oxidants can alter the structure and function of SP-D. Experimental design and results To test our hypothesis, we exposed purified human or rat SP-D to HOC1 or MPO System (MPO+H2O2+Chloride ions). Next, the activity of treated SP-D was examined using two well-characterized in vitro assays: bacterial agglutination and binding to solid phase yeast mannan Both HOC1 and MPO System abrogated SP-Ds ability to aggregate bacteria and decreased its ability to bind to mannan. Additional studies using mutant SP-D suggest oxidative inactivation of SP-D C-type lectin region, the carbohydrate recognition domain (CRD). In contrast to HOC1, H2O2 had no effect on SP-D function at ail concentrations examined. Moreover, electrophoretic analysis of HOCl-treated SP-D demonstrated high molecular weight complexes by comparison to control SP-D. Conclusion Our data suggest that MPO-derived oxidants such as HOC1 target SP-D within its CRD region and broadly interferes with its function(s). Studies are underway to investigate the mechanism(s) of oxidative inactivation of SP-D.