Mutagenesis of surfactant protein D informed by evolution and x-ray crystallography enhances defenses against influenza A virus in vivo

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.     

Interaction of recombinant surfactant protein D with lipopolysaccharide: conformation and orientation of bound protein by IRRAS and simulations

Effective innate host defense requires early recognition of pathogens. Surfactant protein D (SP-D), shown to play a role in host defense, binds to the lipopolysaccharide (LPS) component of Gram-negative bacterial membranes. Binding takes place via the carbohydrate recognition domain (CRD) of SP-D. Recombinant trimeric neck+CRDs (NCRD) have proven valuable in biophysical studies of specific interactions. Although X-ray crystallography has provided atomic level information on NCRD binding to carbohydrates and other ligands, molecular level information about interactions between SP-D and biological ligands under physiologically relevant conditions is lacking. Infrared reflection-absorption spectroscopy (IRRAS) provides molecular structure information from films at the air/water interface where protein adsorption to LPS monolayers serves as a model for protein-lipid interaction. In the current studies, we examine the adsorption of NCRDs to Rd 1 LPS monolayers using surface pressure measurements and IRRAS. Measurements of surface pressure, Amide I band intensities, and LPS acyl chain conformational ordering, along with the introduction of EDTA, permit discrimination of Ca (2+)-mediated binding from nonspecific protein adsorption. The findings support the concept of specific binding between the CRD and heptoses in the core region of LPS. In addition, a novel simulation method that accurately predicts the IR Amide I contour from X-ray coordinates of NCRD SP-D is applied and coupled to quantitative IRRAS equations providing information on protein orientation. Marked differences in orientation are found when the NCRD binds to LPS compared to nonspecific adsorption. The geometry suggests that all three CRDs are simultaneously bound to LPS under conditions that support the Ca (2+)-mediated interaction.     

Recognition of heptoses and the inner core of bacterial lipopolysaccharides by surfactant protein d

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-alpha-d-manno-heptose (l,d-heptose), but not by 3-deoxy-alpha-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 alpha1-->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-alpha-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.     

Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.     

Critical role for cross-linking of trimeric lectin domains of surfactant protein D in antiviral activity against influenza A virus

Collectins are multimeric host defence lectins with trimeric CRDs (carbohydrate-recognition domains) and collagen and N-terminal domains that form higher-order structures composed of four or more trimers. Recombinant trimers composed of only the CRD and adjacent neck domain (termed NCRD) retain binding activity for some ligands and mediate some functional activities. The lung collectin SP-D (surfactant protein D) has strong neutralizing activity for IAVs (influenza A viruses) in vitro and in vivo, however, the NCRD derived from SP-D has weak viral-binding ability and lacks neutralizing activity. Using a panel of mAbs (monoclonal antibodies) directed against the NCRD in the present study we show that mAbs binding near the lectin site inhibit antiviral activity of full-length SP-D, but mAbs which bind other sites on the CRD do not. Two of the non-blocking mAbs significantly increased binding and antiviral activity of NCRDs as assessed by haemagglutination and neuraminidase inhibition and by viral neutralization. mAb-mediated cross-linking also enabled NCRDs to induce viral aggregation and to increase viral uptake by neutrophils and virus-induced respiratory burst responses by these cells. These results show that antiviral activities of SP-D can be reproduced without the N-terminal and collagen domains and that cross-linking of NCRDs is essential for antiviral activity of SP-D with respect to IAV.     

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.     

Arg343 in human surfactant protein D governs discrimination between glucose and N-acetylglucosamine ligands

Surfactant protein D (SP-D), one of the members of the collectin family of C-type lectins, is an important component of pulmonary innate immunity. SP-D binds carbohydrates in a calcium-dependent manner, but the mechanisms governing its ligand recognition specificity are not well understood. SP-D binds glucose (Glc) stronger than N-acetylglucosamine (GlcNAc). Structural superimposition of hSP-D with mannose- binding protein C (MBP-C) complexed with GlcNAc reveals steric clashes between the ligand and the side chain of Arg343 in hSP-D. To test whether Arg343 contributes to Glc > GlcNAc recognition specificity, we constructed a computational model of Arg343-->Val (R343V) mutant hSP-D based on homology with MBP-C. Automated docking of alpha-Me-Glc and alpha-Me-GlcNAc into wild-type hSP-D and the R343V mutant of hSP-D suggests that Arg343 is critical in determining ligand-binding specificity by sterically prohibiting one binding orientation. To empirically test the docking predictions, an R343V mutant recombinant hSP-D was constructed. Inhibition analysis shows that the R343V mutant binds both Glc and GlcNAc with higher affinity than the wild-type protein and that the R343V mutant binds Glc and GlcNAc equally well. These data demonstrate that Arg343 is critical for hSP-D recognition specificity and plays a key role in defining ligand specificity differences between MBP and SP-D. Additionally, our results suggest that the number of binding orientations contributes to monosaccharide binding affinity.     

Human surfactant protein D (SP-D) binds Mycoplasma pneumoniae by high affinity interactions with lipids

Increasing evidence now identifies surfactant protein D (SP-D) as an important element of the innate immune system of the lung. In this study, we examined the interactions of rat and human SP-D with the human pathogen, Mycoplasma pneumoniae. Rat and human SP-D bound the organism with high affinity in a reaction that required Ca(2+) and was inhibited by EGTA. Membranes derived from the organism bound the proteins in a similar manner, except the rat SP-D also exhibited a significant level of Ca(2+)-independent binding. Pretreatment of membranes with proteases did not alter the Ca(2+)-dependent SP-D binding of membranes by either protein. Mannose, glucose, maltose, and inositol, at millimolar concentrations, competed for human SP-D binding to the bacterial membrane. Lipids extracted from membranes and separated by two-dimensional thin layer chromatography bound human SP-D with high affinity in a Ca(2+)-dependent reaction. A tandem mutant of SP-D with E321Q and N323D substitutions, failed to bind M. pneumoniae lipids, directly implicating the carbohydrate recognition domain in the interaction. The interaction of rat and human SP-D with M. pneumoniae was unaffected by the presence of surfactant lipids and the hydrophobic surfactant proteins. These findings demonstrate that M. pneumoniae is likely to be recognized by SP-D in the alveolar environment and that primary determinants recognized on the organism are lipid components of the cell membrane.     

The amino-terminal heptad repeats of the coiled-coil neck domain of pulmonary surfactant protein d are necessary for the assembly of trimeric subunits and dodecamers

Pulmonary surfactant protein D (SP-D), a lung host defense protein, is assembled as multimers of trimeric subunits. Trimerization of SP-D monomers is required for high affinity saccharide binding, and the oligomerization of trimers is required for many of its functions. A peptide containing the alpha-helical neck region can spontaneously trimerize in vitro. However, it is not known whether this sequence is necessary for the complete cellular assembly of disulfide-cross-linked, trimeric subunits and dodecamers. For the present studies, we synthesized mutant cDNAs with deletions or site-directed substitutions in the neck domain of rat SP-D, and examined the assembly of the newly synthesized proteins after transfection of CHO-K1 cells. The neck domain contains three "classical" heptad repeat motifs with leucine residues at the "d position," and a distinctive C-terminal repeat previously suggested to drive trimeric chain association. Deletion of the highly conserved core of the latter repeat (FSRYLKK) did not interfere with the secretion of dodecamers with lectin activity. By contrast, deletion of the entire neck domain or deletion of one or two amino-terminal repeats resulted in defective molecular assembly. The secreted proteins eluted in the position of monomers by gel filtration under nondenaturing conditions. In addition, the neck + carbohydrate recognition domain of SP-D was necessary and sufficient for the trimerization of a heterologous collagen sequence located amino-terminal to the trimeric coiled-coil. These studies provide strong evidence that the amino-terminal heptad repeats of the neck domain are necessary for the intracellular, trimeric association of SP-D monomers and for the assembly and secretion of functional dodecamers.     

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-alpha-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.     

Induction of macrophage matrix metalloproteinase biosynthesis by surfactant protein D

Recent studies strongly suggest that surfactant protein D (SP-D) plays important roles in pulmonary host defense and the regulation of immune and inflammatory reactions in the lung. Although SP-D can bind to alveolar macrophages and can elicit their chemotaxis, relatively little is known about the direct cellular consequences of SP-D on the function of these cells. Because matrix metalloproteinases (MMPs) are synthesized in increased amounts in response to various proinflammatory stimuli, we investigated the capacity of SP-D to modulate the production of MMPs by freshly isolated human alveolar macrophages. Unexpectedly we found that recombinant rat SP-D dodecamers selectively induce the biosynthesis of collagenase-1 (MMP-1), stromelysin (MMP-3), and macrophage elastase (MMP-12) without significantly increasing the production of tumor necrosis factor alpha and interleukin-1beta. SP-D did not alter the production of these MMPs by fibroblasts. Phosphatidylinositol, a surfactant-associated ligand that interacts with the carboxyl-terminal neck and carbohydrate recognition domains of SP-D, inhibited the SP-D-dependent increase in MMP biosynthesis. A trimeric, recombinant protein consisting of only the neck and carbohydrate recognition domain did not augment metalloproteinase production, suggesting that the stimulatory effect on MMP production depends on an appropriate spatial presentation of trimeric lectin domains. Although SP-D dodecamers can selectively augment metalloproteinase activity in vitro, this effect may be competitively inhibited by tissue inhibitors of metalloproteinases or surfactant-associated ligands in vivo.     

The major glycolipid recognized by SP-D in surfactant is phosphatidylinositol.

Surfactant protein D (SP-D), a multimeric calcium-dependent lectin isolated from pulmonary alveolar lavage, has been previously shown to interact reversibly with crude surfactant [Persson et al. (1990) J. Biol. Chem. 265, 5755-5760]. In this study, SP-D is shown to interact reversibly with a preparation of organelles enriched in lamellar bodies, in a manner inhibited by calcium-chelating agents and by competing saccharides. An interaction with an endogenous glycoprotein could not be identified by electrophoresis of surfactant or lamellar body-associated proteins followed by electrotransfer of the separated proteins to nitrocellulose and then probing with radioiodinated SP-D via lectin overlay. Separation of the surfactant or lamellar body lipids on two-dimensional thin-layer chromatography (2D-TLC) followed by probing with radioiodinated SP-D via lectin overlay demonstrated binding to a single lipid. This interaction was dependent on the presence of calcium and was inhibited by competing saccharides. By assaying column fractions for the ability to bind radioiodinated SP-D after TLC, the glycolipid was purified to homogeneity and identified as phosphatidylinositol (PI). Identification was confirmed by mass spectrometry. We further demonstrate the ability of radiolabeled SP-D to bind to PI presented in a lipid bilayer through separation of free SP-D from liposome-bound SP-D on density gradients of Percoll. The interaction of SP-D with PI is dependent on calcium and inhibited by competing saccharides. SP-D binds with similar efficiency to liposomes with mole fractions of PI ranging from 2.5% to 30%, thereby demonstrating the lectin's ability to recognize mole fractions of PI available in surfactant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.     

High-resolution structural insights into ligand binding and immune cell recognition by human lung surfactant protein D

Lung surfactant protein D (SP-D) can directly interact with carbohydrate residues on pulmonary pathogens and allergens, stimulate immune cells, and manipulate cytokine and chemokine profiles during the immune response in the lungs. Therapeutic administration of rfhSP-D, a recombinant homotrimeric fragment of human SP-D comprising the alpha-helical coiled-coil neck plus three CRDs, protects mice against lung allergy and infection caused by the fungal pathogen Aspergillus fumigatus. The high resolution crystal structures of maltose-bound rfhSP-D to 1.4A, and of rfhSP-D to 1.6A, define the fine detail of the mode and nature of carbohydrate recognition and provide insights into how a small fragment of human SP-D can bind to allergens/antigens or whole pathogens, and at the same time recruit and engage effector cells and molecules of humoral immunity. A previously unreported calcium ion, located on the trimeric axis in a pore at the bottom of the funnel formed by the three CRDs and close to the neck-CRD interface, is coordinated by a triad of glutamate residues which are, to some extent, neutralised by their interactions with a triad of exposed lysine residues in the funnel. The spatial relationship between the neck and the CRDs is maintained internally by these lysine residues, and externally by a glutamine, which forms a pair of hydrogen-bonds within an external cleft at each neck-CRD interface. Structural links between the central pore and the cleft suggest a possible effector mechanism for immune cell surface receptor binding in the presence of bound, extended natural lipopolysaccharide and phospholipid ligands. The structural requirements for such an effector mechanism, involving both the trimeric framework for multivalent ligand binding and recognition sites formed from more than one subunit, are present in both native hSP-D and rfhSP-D, providing a possible explanation for the significant biological activity of rfhSP-D.     

Structural analysis of the human galectin-9 N-terminal carbohydrate recognition domain reveals unexpected properties that differ from the mouse orthologue

Galectins are a family of β-galactoside-binding lectins that contain a conserved carbohydrate recognition domain (CRD). They exhibit high affinities for small β-galactosides as well as variable binding specificities for complex glycoconjugates. Structural and biochemical analyses of the mechanism governing specific carbohydrate recognition provide a useful template to elucidate the function of these proteins. Here we report the crystal structures of the human galectin-9 N-terminal CRD (NCRD) in the presence of lactose and Forssman pentasaccharide. Mouse galectin-9 NCRD, the structure of which was previously solved by our group, forms a non-canonical dimer in both the crystal state and in solution. Human galectin-9 NCRD, however, exists as a monomer in crystals, despite a high sequence identity to the mouse homologue. Comparative frontal affinity chromatography analysis of the mouse and human galectin-9 NCRDs revealed different carbohydrate binding specificities, with disparate affinities for complex glycoconjugates. Human galectin-9 NCRD exhibited a high affinity for Forssman pentasaccharide; the association constant for mouse galectin-9 NCRD was 100-fold less than that observed for the human protein. The combination of structural data with mutational studies demonstrated that non-conserved amino acid residues on the concave surface were important for determination of target specificities. The human galectin-9 NCRD exhibited greater inhibition of cell proliferation than the mouse NCRD. We discuss the biochemical and structural differences between highly homologous proteins from different species.     

Complementation of pulmonary abnormalities in SP-D(-/-) mice with an SP-D/conglutinin fusion protein

Surfactant protein D (SP-D) and serum conglutinin are closely related members of the collectin family of host defense lectins. Although normally synthesized at different anatomic sites, both proteins participate in the innate immune response to microbial challenge. To discern the roles of specific domains in the function of SP-D in vivo, a fusion protein (SP-D/Cong(neck+CRD)) consisting of the NH(2)-terminal and collagenous domains of rat SP-D (rSP-D) and the neck and carbohydrate recognition domains (CRDs) of bovine conglutinin (Cong) was expressed in the respiratory epithelium of SP-D gene-targeted (SP-D(-/-)) mice. While SP-D/Cong(neck+CRD) fusion protein did not affect lung morphology and surfactant phospholipid levels in the lungs of wild type mice, the chimeric protein substantially corrected the increased lung phospholipids in SP-D(-/-) mice. The SP-D/Cong(neck+CRD) fusion protein also completely corrected defects in influenza A clearance and inhibited the exaggerated inflammatory response that occurs following viral infection. However, the chimeric protein did not ameliorate the ongoing lung inflammation, enhanced metalloproteinase expression, and alveolar destruction that characterize this model of SP-D deficiency. By contrast, a single arm mutant (RrSP-D(Ser15,20)) partially restored antiviral activity but otherwise failed to rescue the deficient phenotype. Our findings directly implicate the CRDs of both SP-D and conglutinin in host defense in vivo. Our findings also strongly suggest that the molecular mechanisms underlying impaired pulmonary host defense and abnormal lipid metabolism are distinct from those that promote ongoing inflammation and the development of emphysema.     

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

Pigs can act as intermediate hosts by which reassorted influenza A virus (IAV) strains can be transmitted to humans and cause pandemic influenza outbreaks. The innate host defense component surfactant protein D (SP-D) interacts with glycans on the hemagglutinin of IAV and contributes to protection against IAV infection in mammals. This study shows that a recombinant trimeric neck lectin fragment derived from porcine SP-D (pSP-D) exhibits profound inhibitory activity against IAV, in contrast to comparable fragments derived from human SP-D. Crystallographic analysis of the pSP-D fragment complexed with a viral sugar component shows that a unique tripeptide loop alters the lectin site conformation of pSP-D. Molecular dynamics simulations highlight the role of this flexible loop, which adopts a more stable conformation upon sugar binding and may facilitate binding to viral glycans through contact with distal portions of the branched mannoside. The combined data demonstrate that porcine-specific structural features of SP-D contribute significantly to its distinct anti-IAV activity. These findings could help explain why pigs serve as important reservoirs for newly emerging pathogenic IAV strains.     

Importance of the carboxy-terminal 25 amino acid residues of lung collectins in interactions with lipids and alveolar type II cells

Surfactant proteins A and D (SP-A and SP-D) are structurally related members of the collectin family found in the alveolar compartment of the lung. SP-A binds dipalmitoylphosphatidylcholine (DPPC) and galactosylceramide (GalCer), induces liposome aggregation, and regulates the uptake and secretion of surfactant lipids by alveolar type II cells in vitro. SP-D binds phosphatidylinositol (PI) and glucosylceramide. The purpose of this study was to identify a critical stretch of primary sequence in the SP-A region Cys(204)-Phe(228) and the SP-D region Cys(331)-Phe(355) that is involved in protein-specific lipid and type II cell interactions. Chimeras ad1 and ad2 were constructed with rat SP-A/SP-D splice junctions at Cys(218)/Gly(346) and Lys(203)/Cys(331), respectively. Chimera ad1 but not ad2 retained DPPC liposome binding activity. Both chimeras retained significant binding to GalCer liposomes. Chimera ad1 did not bind to PI, whereas chimera ad2 acquired a significant PI binding. Both chimeras failed to induce liposome aggregation and to interact with alveolar type II cells. In addition, monoclonal antibody 1D6 that blocks specific SP-A functions did not recognize either chimera. From these results, we conclude that (1) the SP-A region Leu(219)-Phe(228) is required for liposome aggregation and interaction with alveolar type II cells, (2) the SP-A region Cys(204)-Cys(218) is required for DPPC binding, (3) the SP-D region Cys(331)-Phe(355) is essential for minimal PI binding, and (4) the epitope for mAb 1D6 is located at the region contiguous to the SP-A region Leu(219)-Phe(228).     

Characterization of pulmonary surfactant protein D: its copurification with lipids

Surfactant protein D (SP-D) is a collagenous surfactant associated protein synthesized by alveolar type II cells. SP-D was purified from the supernatant of rat bronchoalveolar lavage fluids obtained by centrifugation at 33,000 x gav for 16 h. The contents of SP-D and SP-A in fractions obtained by the centrifugation of rat bronchoalveolar lavage were determined by enzyme-linked immunoassay. The total content of SP-D was approximately 12% of that of SP-A in these lavage fluids. 99.1% of SP-A was present in the 33,000g pellet, whereas 71.1% of SP-D was in the 33,000g supernatant. Analysis by high performance liquid chromatography reveals that lipids are copurified with isolated SP-D. Phosphatidylcholine accounted for 84.8% of the phospholipids copurified with SP-D. Unlike SP-A, SP-D in the purified and delipidated form failed to compete with 125I-labeled SP-A for phosphatidylcholine binding, and to aggregate phospholipid liposomes. The present study demonstrates that lipids are copurified with SP-D, that SP-D and SP-A distribute differently in rat bronchoalveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fashion as SP-A.     

Surfactant protein D binds to Mycobacterium tuberculosis bacilli and lipoarabinomannan via carbohydrate-lectin interactions resulting in reduced phagocytosis of the bacteria by macrophages.

Surfactant protein-D (SP-D) is a collectin produced in the distal lung airspaces that is believed to play an important role in innate pulmonary immunity. Naive immunologic responses to Mycobacterium tuberculosis (M.tb) are especially important in the lung, since entry of this inhaled pathogen into the alveolar macrophage is a pivotal event in disease pathogenesis. Here we investigated SP-D binding to M.tb and the effect of this binding on the adherence of M. tb to human macrophages. These studies demonstrate specific binding of SP-D to M.tb that is saturable, calcium dependent, and carbohydrate inhibitable. In addition to purified SP-D, SP-D in bronchoalveolar lavage fluids from healthy donors and patients with alveolar proteinosis also binds to M.tb. Incubation of M.tb with SP-D results in agglutination of the bacteria. In contrast to its binding to M.tb, SP-D binds minimally to the avirulent Mycobacterium smegmatis. SP-D binds predominantly to lipoarabinomannan from the virulent Erdman strain of M.tb, but not the lipoarabinomannan from M. smegmatis. The binding of SP-D to Erdman lipoarabinomannan is mediated by the terminal mannosyl oligosaccharides of this lipoglycan. Incubation of M.tb with subagglutinating concentrations of SP-D leads to reduced adherence of the bacteria to macrophages (62.7% of control adherence +/- 3.3% SEM, n = 8), whereas incubation of bacteria with surfactant protein A leads to significantly increased adherence to monocyte-derived macrophages. These data provide evidence for specific binding of SP-D to M. tuberculosis and indicate that SP-D and surfactant protein A serve different roles in the innate host response to this pathogen in the lung.