Rat Surfactant Protein D Research Articles

Structure binding relationship of human surfactant protein D and various lipopolysaccharide inner core structures.

As a major player of the innate immune system, surfactant protein D (SP-D) recognizes and promotes elimination of various pathogens such as Gram-negative bacteria. SP-D binds to l-glycero-d-manno-heptose (Hep), a constituent of the partially conserved lipopolysaccharide (LPS) inner core of many Gram-negative bacteria. Binding and affinity of trimeric human SP-D to Hep in distinct LPS inner core glycans differing in linkages and adjacent residues was elucidated using glycan array and surface plasmon resonance measurements that were compared to in silico interaction studies. The combination of in vitro assays using defined glycans and molecular docking and dynamic simulation approaches provides insights into the interaction of trimeric SP-D with those glycan ligands. Trimeric SP-D wildtype recognized larger LPS inner core oligosaccharides with slightly enhanced affinity than smaller compounds suggesting the involvement of stabilizing secondary interactions. A trimeric human SP-D mutant D324N+D325N+R343K resembling rat SP-D bound to various LPS inner core structures in a similar pattern as observed for the wildtype but with higher affinity. The selective mutation of SP-D promotes targeting of LPS inner core oligosaccharides on Gram-negative bacteria to develop novel therapeutic agents.

Increased Surfactant Protein D Fails to Improve Bacterial Clearance and Inflammation in serpinB1−/− Mice

Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis.

Surfactant protein D modulates levels of IL-10 and TNF-α in intrauterine compartments during lipopolysaccharide-induced preterm birth

Prematurity is the main cause of perinatal mortality and morbidity, and preterm birth is often associated with intrauterine inflammation. Surfactant protein D (SP-D) functions in lung homeostasis and has multiple roles in innate immunity. It is present in amniotic fluid and in gestational tissues. We propose that SP-D may regulate intrauterine inflammatory responses related to preterm labor. Our aim was to investigate the role of SP-D in lipopolysaccharide-induced preterm birth in mice that overexpress rat SP-D (rSP-D) under the human SP-C promoter. SP-D protein in amniotic fluid and in gestational tissues was detected by western analysis. TNF-α, IL-10, and IL-6 concentrations in serum and amniotic fluid and mRNA levels in gestational tissues were quantified using cytometric bead array and ribonuclease protection assay, respectively. Increased levels of SP-D protein were detected in the amniotic fluid and gestational tissues of rSP-D mice. Lipopolysaccharide given at 17days post-coitum to rSP-D dams led to preterm birth of live-born offspring within 18h. Preterm birth of live-born pups was induced with a lower dose of lipopolysaccharide compared to wild-type mice. In rSP-D mice, the lipopolysaccharide-induced levels of TNF-α and IL-10 in amniotic fluid and fetal serum and the expression of IL-10 in placenta and fetal membranes were significantly different from wild-type mice. We conclude that SP-D in fetal and gestational tissues modulates the levels of intrauterine inflammatory mediators involved in preterm birth and may contribute to inflammatory processes related to spontaneous preterm labor.

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.

Surfactant protein D modulates pulmonary clearance of pollen starch granules

ABSTRACTPollen starch granules (PSGs) are allergen particles that get into contact with pulmonary surfactant and phagocytes in the terminal airways. In this study, the effects of surfactant protein D (SP-D) on the interaction of PSGs with phagocytes and on the pulmonary clearance of PSGs were determined. Fluorescently labeled PSGs were incubated in vitro with murine lung macrophages or dendritic cells (DCs) ± recombinant rat SP-D (rrSP-D). In addition, the effect of SP-D on uptake of PSGs by lung macrophages and DCs was studied in vivo. Furthermore, PSGs were instilled in Balb/c mice and the effects of SP-D on total lung clearance were assessed by optical imaging. SP-D treatment increased the number of PSG-positive macrophages and DCs in vitro. Furthermore, SP-D accelerated uptake/binding by alveolar macrophages and reduced the number of PSG-positive tissue macrophages and DCs at 24 hours. However, SP-D did not affect total lung clearance of PSGs and it did not enhance the T-cell proliferation induced by PSG-positive DCs. In conclusion, SP-D increased PSG-positive cells in vitro and accelerated PSG binding/uptake in vivo. The observed effects were limited to cellular clearance mechanisms and did not affect the total clearance of PSGs from the lung.

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-H(2)O(2)-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.

Surfactant Protein D Protects against Acute Hyperoxic Lung Injury

Surfactant protein D (SP-D) is a member of the collectin family of soluble, innate, host defense molecules with demonstrated immunomodulatory properties in vitro. Constitutive absence of SP-D in mice is associated with lung inflammation, alteration in surfactant lipid homeostasis, and increased oxidative-nitrative stress. To test the hypothesis that SP-D would protect against acute lung injury from hyperoxia in vivo. Transgenic mice overexpressing rat SP-D constitutively (SP-D OE) or conditionally via regulation with doxycycline (SP-D Dox-on) were subjected to continuous hyperoxic challenge for up to 14 days. Compared with littermate control mice (wild-type [WT]), SP-D OE mice exposed to 80% O(2) demonstrated substantially increased survival accompanied by significant reductions in wet to dry lung ratios and bronchoalveolar lavage (BAL) protein. Although SP-D OE and WT mice exhibited a twofold increase in total BAL cells and neutrophilia in response to hyperoxia, the SP-D OE group had lower levels of BAL proinflammatory cytokines and chemokines, including IL-6, tumor necrosis factor-alpha, and monocyte chemotactic protein-1; increased mRNA levels of the transcription factor NF-E2 related factor-2 (NRF-2) and phase 2 antioxidants hemoxygenase-1 (HO-1), glutathione peroxidase-2 (GPx-2) and NAD(P)H quinone oxidoreductase-1 (Nqo-1); and decreases in lung tissue thiobarbituric acid-reactive substances. As proof of principle, the protective role of SP-D on hyperoxic injury was confirmed as SP-D Dox-on mice exposed to 85% O(2) demonstrated increased mortality upon withdrawal of doxycycline. Local expression of SP-D protects against hyperoxic lung injury through modulation of proinflammatory cytokines and antioxidant enzymatic scavenger systems.

Critical Role of Arg/Lys343 in the Species-Dependent Recognition of Phosphatidylinositol by Pulmonary Surfactant Protein D,

Surfactant protein D (SP-D) plays important roles in lung host defense. However, it can also recognize specific host molecules and contributes to surfactant homeostasis. The major known surfactant-associated ligand is phosphatidylinositol (PI). Trimeric neck-carbohydrate recognition domains (NCRDs) of rat and human SP-D exhibited dose-dependent, calcium-dependent, and inositol-sensitive binding to solid-phase PI and to multilamellar PI liposomes. However, the rat protein exhibited a >5-fold higher affinity for solid-phase PI than the human NCRD. In addition, human dodecamers, but not full-length human trimers, efficiently coprecipitated with multilamellar PI liposomes in the presence of calcium. A human NCRD mutant resembling the rat and mouse proteins at position 343 (hR343K) showed much stronger binding to PI. A reciprocal rat mutant with arginine at the position of lysine 343 (rK343R) showed weak binding to PI, even weaker than that of the wild-type human protein. Crystal complexes of the human trimeric NCRD with myoinositol and inositol 1-phosphate showed binding of the equatorial OH groups of the cyclitol ring of the inositol to calcium at the carbohydrate binding site. Myoinositol binding occurred in two major orientations, while inositol 1-phosphate appeared primarily constrained to a single, different orientation. Our studies directly implicate the CRD in PI binding and reveal unexpected species differences in PI recognition that can be largely attributed to the side chain of residue 343. In addition, the studies indicate that oligomerization of trimeric subunits is an important determinant of recognition of PI by human SP-D.

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-D's 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.

Purification, characterization and immunolocalization of porcine surfactant protein D

Surfactant protein D (SP-D) is a collectin believed to play an important role in innate immunity. SP-D is characterized by having a collagen-like domain and a carbohydrate recognition domain (CRD), which has a specific Ca(2+)-dependent specificity for saccharides and thus the ability to bind complex glycoconjugates on micro-organisms. This paper describes the tissue immunolocalization of porcine SP-D (pSP-D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP-D. Porcine SP-D was purified from porcine bronchoalveolar lavage (BAL) by maltose-agarose and immunoglobulin M affinity chromatography. The purified protein appeared on sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a band of approximately 53,000 MW in the reduced state and approximately 138,000 MW in the unreduced state. Porcine SP-D was sensitive to collagenase digestion and N-deglycosylation, which reduced the molecular mass to approximately 24,000 MW and approximately 48,000 MW respectively, in the reduced state. N-deglycosylation of the collagen-resistant fragment, reduced the molecular mass to approximately 21,000 MW showing the presence of an N-glycosylation site located in the CRD. Porcine SP-D bound to solid-phase mannan in a dose and Ca(2+)-dependent manner with a saccharide specificity similar to rat and human SP-D. The purified protein was used for the production of a monoclonal anti-pSP-D antibody. The antibody reacted specifically with pSP-D in the reduced and unreduced state when analysed by Western blotting. Immunohistochemical evaluation of normal porcine tissues showed pSP-D immunoreactivity predominantly in Clara cells and serous cells of the bronchial submucosal glands, and to a lesser extent in alveolar type II cells, epithelial cells of the intestinal glands (crypts of Lieberkuhn) in the duodenum, jejunum and ileum and serous cells of the dorsolateral lacrimal gland.

Neutrophil Serine Proteinases Inactivate Surfactant Protein D by Cleaving within a Conserved Subregion of the Carbohydrate Recognition Domain

Surfactant protein D (SP-D) plays important roles in innate immunity including the defense against bacteria, fungi, and respiratory viruses. Because SP-D specifically interacts with neutrophils that infiltrate the lung in response to acute inflammation and infection, we examined the hypothesis that the neutrophil-derived serine proteinases (NSPs): neutrophil elastase, proteinase-3, and cathepsin G degrade SP-D. All three human NSPs specifically cleaved recombinant rat and natural human SP-D dodecamers in a time- and dose-dependent manner, which was reciprocally dependent on calcium concentration. The NSPs generated similar, relatively stable, disulfide cross-linked immunoreactive fragments of approximately 35 kDa (reduced), and sequencing of a major catheptic fragment definitively localized the major sites of cleavage to a highly conserved subregion of the carbohydrate recognition domain. Cleavage markedly reduced the ability of SP-D to promote bacterial aggregation and to bind to yeast mannan in vitro. Incubation of SP-D with isolated murine neutrophils led to the generation of similar fragments, and cleavage was inhibited with synthetic and natural serine proteinase inhibitors. In addition, neutrophils genetically deficient in neutrophil elastase and/or cathepsin G were impaired in their ability to degrade SP-D. Using a mouse model of acute bacterial pneumonia, we observed the accumulation of SP-D at sites of neutrophil infiltration coinciding with the appearance of approximately 35-kDa SP-D fragments in bronchoalveolar lavage fluids. Together, our data suggest that neutrophil-derived serine proteinases cleave SP-D at sites of inflammation with potential deleterious effects on its biological functions.

Surfactant protein D regulates airway function and allergic inflammation through modulation of macrophage function.

The lung collectin surfactant protein D (SP-D) is an important component of the innate immune response but is also believed to play a role in other regulatory aspects of immune and inflammatory responses within the lung. The role of SP-D in the development of allergen-induced airway inflammation and hyperresponsiveness (AHR) is not well defined. SP-D levels progressively increased up to 48 hours after allergen challenge of sensitized mice and then subsequently decreased. The levels of SP-D paralleled the development of airway eosinophilia and AHR. To determine if this association was functionally relevant, mice were administered rat SP-D (rSP-D) intratracheally. When given to sensitized mice before challenge, AHR and eosinophilia were reduced by rSP-D in a dose-dependent manner but not by mutant rSP-D. rSP-D administration resulted in increased levels of interleukin (IL)-10, IL-12, and IFN-gamma in bronchoalveolar lavage fluid and reduced goblet cell hyperplasia. Culture of alveolar macrophages together with SP-D and allergen resulted in increased production of IL-10, IL-12, and IFN-gamma. These results indicate that SP-D can (negatively) regulate the development of AHR and airway inflammation after airway challenge of sensitized mice, at least in part, by modulating the function of alveolar macrophages.

Proteolysis of surfactant protein D by cystic fibrosis relevant proteases.

Surfactant protein D (SP-D) is an important innate host defense molecule that has been shown to interact with cystic fibrosis (CF)-associated pathogens. Previous studies demonstrated that rat SP-D is highly resistant to degradation by a wide range of proteolytic enzymes. The aim of this study was to examine whether human SP-D can be degraded by CF relevant proteases ex vivo and in vitro. Bronchoalveolar lavage fluids (BALFs) of 11 patients with CF in a stable clinical condition were examined for SP-D by immunoblotting. In vitro, purified human SP-D was treated with human leukocyte elastase, proteinase 3, cathepsin G or Pseudomonas elastase followed by immunoblotting with specific antibodies to SP-D. In BALF of 8 of the 11 patients investigated, proteolytic fragments or absence of SP-D were detected. In vitro proteolysis of SP-D was observed in a time-dependent manner for each protease applied. The presence of Ca(++) at a physiologic concentration delayed, but did not prevent the degradation. We conclude that SP-D is an important target of numerous proteases present in the CF lung. Host defense is probably impaired due to proteolysis of SP-D and may contribute to the suppurative lung disease in CF.

Reversibility of Pulmonary Abnormalities by Conditional Replacement of Surfactant Protein D (SP-D) in Vivo

Surfactant protein D (SP-D) gene-targeted mice develop severe pulmonary disease associated with emphysema, pulmonary lipidosis, and foamy macrophage infiltrations. To determine the potential reversibility of these abnormalities, transgenic mice were developed in which SP-D was conditionally replaced in the respiratory epithelium of SP-D(-/-) mice. SP-D was not detected in the absence of doxycycline. Treatment with doxycycline after birth restored pulmonary SP-D concentrations and corrected pulmonary pathology at adulthood. When SP-D was replaced in adult SP-D(-/-) mice, alveolar SP-D was restored within 3 days, pulmonary lipid abnormalities were corrected, but emphysema persisted. In corrected adult SP-D(-/-) mice, loss of SP-D caused focal emphysema and pulmonary inflammation but did not cause phospholipid abnormalities characteristic of SP-D(-/-) mice. Thus, abnormalities in surfactant phospholipid homeostasis and alveolar macrophage abnormalities were readily corrected by restoration of SP-D. However, once established, emphysema was not reversed by SP-D. SP-D-dependent processes regulating surfactant lipid homeostasis were disassociated from those mediating emphysema.

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.

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.

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.

Activity of Pulmonary Surfactant Protein-D (SP-D) in Vivo Is Dependent on Oligomeric Structure

Pulmonary surfactant protein-D (SP-D) is a member of the collectin family of C-type lectins that is synthesized in many tissues including respiratory epithelial cells in the lung. SP-D is assembled predominantly as dodecamers consisting of four homotrimeric subunits each. Association of these subunits is stabilized by interchain disulfide bonds involving two conserved amino-terminal cysteine residues (Cys-15 and Cys-20). Mutant recombinant rat SP-D lacking these residues (RrSP-Dser15/20) is secreted in cell culture as trimeric subunits rather than as dodecamers. In this study, transgenic mice that express this mutant were generated to elucidate the functional importance of SP-D oligomerization in vivo. Expression of RrSP-Dser15/20 failed to correct the pulmonary phospholipid accumulation and emphysema characteristic of SP-D null (mSP-D-/-) mice. Expression of high concentrations of the mutant protein in wild-type mice reduced the abundance of disulfide cross-linked oligomers of endogenous SP-D in the bronchoalveolar lavage fluid and demonstrated a phenotype that partially overlapped with that of the SP-D-/- mice; the animals developed emphysema and foamy macrophages without the associated abnormalities in alveolar phospholipids typical of SP-D-/- mice. Development of foamy macrophages in SP-D-deficient mice is not secondary to the increased abundance of surfactant phospholipids. Disulfide cross-linked SP-D oligomers are required for the regulation of surfactant phospholipid homeostasis and the prevention of emphysema and foamy macrophages in vivo.

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.

Purification and biochemical characterization of equine pulmonary surfactant protein D

Abstract Objective To characterize surfactant protein D (SP-D) isolated from bronchoalveolar lavage fluid (BALF) of healthy horses. Sample Population BALF from 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) without history or clinical signs of respiratory tract disease. Procedure BALF was obtained and centrifuged at 33,000 × g. The supernatant was applied to a mannose-Sepharose 6B affinity column in the presence of calcium, and the bound protein fraction was analyzed by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis; amino acid composition was determined and partial sequencing was done. Phospholipid binding and liposome aggregation assay were performed, using purified proteins. Results The protein isolated by use of mannose affinity matrices was SP-D. It bound carbohydrates and phosphatidylinositol, which are the characteristic features of SP-D isolated from other animal species. Amino acid analysis and partial primary sequence of the isolated protein indicated high homology with rat and human SP-D. Furthermore, immunoblot analysis indicated that equine SP-D reacted with human and rat SP-D-specific antibodies. Conclusion and Clinical Relevance SP-D exists in equine lungs; its measurement may be useful in evaluating equine lung disease. (Am J Vet Res 1999;60:368–372)