The major lung surfactant protein, SP 28-36, is a calcium-dependent, carbohydrate-binding protein

SP 28-36, a major protein of pulmonary surfactant, has striking amino acid sequence homology with soluble mannose-binding proteins isolated from rat liver and contains residues common to the carbohydrate-binding domains of other mammalian lectins. We have used carbohydrate-affinity chromatography to investigate carbohydrate-binding properties of SP 28-36 isolated from canine and human (alveolar proteinosis patients) lung lavage. SP 28-36 binds to immobilized D-mannose, L-fucose, D-galactose, and D-glucose. The protein binds only weakly to N-acetyl-D-galactosamine and N acetyl-D-glucosamine. Binding is Ca2+-dependent. The threshold Ca2+ concentration is 0.6 mM and maximal binding occurs with 1 mM Ca2+. Bound protein is quantitatively recovered by elution with 2 mM EDTA. Ba2+, Sr2+, and Mn2+, but not Mg2+, can substitute for Ca2+. Unlike some other mammalian lectins, SP 28-36 binds to carbohydrate at pH 5.0. Recombinant human SP 28-36 isolated from the media of Chinese hamster ovary cells, transfected with a DNA construct encoding SP 28-36, has similar carbohydrate-binding activity to the native proteins. Mannose affinity chromatography of the culture medium of Chinese hamster ovary cells results in an efficient purification of the secreted recombinant human SP 28-36.     

Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A(2)) and SP-A2 (1A(0)), and the coexpressed SP-A1/SP-A2 (6A(2)/1A(0)) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A(0)) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A(2)) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A(2)/1A(0)) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.     

Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A²) and SP-A2 (1A⁰), and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A⁰) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A²) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.     

Binding of surfactant protein A (SP-A) to herpes simplex virus type 1-infected cells is mediated by the carbohydrate moiety of SP-A.

Pulmonary surfactant protein A (SP-A) has been shown to act as an opsonin in the phagocytosis of viruses by alveolar macrophages. To determine whether SP-A binds to viral proteins and which part of the SP-A molecule is involved in this interaction, binding studies were undertaken. SP-A was labeled with fluorescein isothiocyanate, and its binding to herpes simplex virus type 1-infected HEp-2 cells, as a model for virus-infected cells in general, was studied using flow cytometry. The binding of SP-A to virus-infected cells was saturable, reversible, and both time- and concentration-dependent, reaching a maximal level after 30 min at an SP-A concentration of 10 micrograms/ml. An approximately 4-fold increase in binding of SP-A to infected cells over control cells was observed. Yeast mannan, a mannose homopolysaccharide, did not influence the binding. However, heparin inhibited binding of SP-A in a concentration-dependent manner. In addition, heparin could also dissociate cell-bound SP-A, indicating that polyanionic oligosaccharides are involved in the binding of SP-A to virus-infected cells. Deglycosylated SP-A, obtained by digestion with N-glycosidase F, did not bind to infected cells. Heparin or deglycosylation of SP-A had no effect on the stimulation of alveolar macrophages by SP-A. It is concluded that the carbohydrate moiety of SP-A is involved in the recognition of viruses by SP-A and may play a role in the antiviral defenses of the lung.     

Aerosolized endotoxin is immediately bound by pulmonary surfactant protein D in vivo.

Collectins are carbohydrate binding proteins that are implicated in innate host defense. The lung collectins, surfactant proteins A and D (SP-A and SP-D), bind a variety of pathogens in vitro and influence phagocytosis by alveolar macrophages. In this report we show that SP-D binds endotoxin (lipopolysaccharide, LPS) in vivo in a rat model of acute respiratory distress syndrome (ARDS). Intratracheal aerosolization of LPS in rats resulted in the typical features of human ARDS. Total amounts of SP-D, as well as the carbohydrate binding properties of SP-D were measured in lung lavage as a function of time. The amount of SP-D did not change during 24 h. Interestingly, SP-D in lung lavage isolated from rats during the first 2 h after LPS treatment, was not able to bind to carbohydrate. Further analysis revealed that the carbohydrate binding sites of SP-D were occupied by LPS, suggesting that SP-D is an LPS scavenging molecule in vivo. Electron microscopic analysis indicated that, 1 h after LPS aerosolization, aggregates of SP-D with LPS were found in lysosomal structures in alveolar macrophages. We conclude that the lung collectin SP-D binds inhaled endotoxin in vivo, which may help to protect the lung from endotoxin-induced disease.     

Introduction of mannose binding protein-type phosphatidylinositol recognition into pulmonary surfactant protein A.

Pulmonary surfactant protein A (SP-A) and mannose-binding protein A (MBP-A) are collectins in the C-type lectin superfamily. These collectins exhibit unique lipid binding properties. SP-A binds to dipalmitoyl phosphatidylcholine (DPPC) and galactosylceramide (GalCer) and MBP-A binds to phosphatidylinositol (PI). SP-A also interacts with alveolar type II cells. Monoclonal antibodies (mAbs PE10 and PC6) that recognize human SP-A inhibit the interactions of SP-A with lipids and alveolar type II cells. We mapped the epitopes for anti-human SP-A mAbs by a phage display peptide library. Phage selected by mAbs displayed the consensus peptide sequences that are nearly identical to 184TPVNYTNWYRG194 of human SP-A. The synthetic peptide GTPVNYTNWYRG completely blocked the binding of mAbs to human SP-A. Chimeric proteins were generated in which the rat SP-A region Thr174-Gly194 or the human SP-A region Ser174-Gly194 was replaced with the MBP-A region Thr164-Asp184 (rat ama4 or hu ama4, respectively). The mAbs failed to bind hu ama4. Rat ama4 bound to an affinity matrix on mannose-sepharose but lost all of the SP-A functions except carbohydrate binding and Ca2+-independent GalCer binding. Strikingly, the rat ama4 chimera acquired the PI binding property that MBP-A exhibits. This study demonstrates that the amino acid residues 174-194 of SP-A and the corresponding region of MBP-A are critical for SP-A-type II cell interaction and Ca2+-dependent lipid binding of collectins.     

Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids.

Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem. 262, 13877-13880). Chromatogram overlays in conjunction with in situ liquid secondary ion mass spectrometry (TLC-LSIMS) of several purified glycosphingolipids and neoglycolipids as well as binding assays with glycolipids immobilized on plastic wells, demonstrate recognition of galactose (human and dog SP-A), glucose, and lactose (human SP-A) in association with specific lipids. In addition, the occurrence of several neutral and acidic glycosphingolipids in human and rat extracellular surfactants and rat alveolar type II cells is described. Selected components among the neutral glycolipids are bound by radiolabeled human SP-A; these are identified by TLC-LSIMS as predominantly ceramide mono- and disaccharides (human surfactant) and ceramide tri- and tetrasaccharides (rat surfactant and type II cells). A recombinant carbohydrate recognition domain (CRD) of human SP-A inhibits the binding of human SP-A to galactosyl ceramide and to galactose- and mannose-bovine serum albumin, indicating that the CRD is directly involved in the binding of SP-A to these ligands. These results provide evidence for a novel type of binding specificity for proteins that have Ca(2+)-dependent CRDs and raise the possibility that glycosphingolipids are endogenous ligands for SP-A.     

Impact of ozone exposure on the phagocytic activity of human surfactant protein A (SP-A) and SP-A variants

Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. SP-A1 and SP-A2 encode human (h) SP-A; SP-A2 products enhance phagocytosis more than SP-A1. Oxidation can affect SP-A function. We hypothesized that in vivo and in vitro ozone-induced oxidation of SP-A (as assessed by its carbonylation level) negatively affects its function in phagocytosis (as assessed by bacteria cell association). To test this, we used P. aeruginosa, rat alveolar macrophages (AMs), hSP-As with varying levels of in vivo (natural) oxidation, and ozone-exposed SP-A2 (1A, 1A0) and SP-A1 (6A2, 6A4) variants. SP-A oxidation levels (carbonylation) were measured; AMs were incubated with bacteria in the presence of SP-A, and the phagocytic index was calculated. We found: 1) the phagocytic activity of hSP-A is reduced with increasing levels of in vivo SP-A carbonylation; 2) in vitro ozone exposure of hSP-A decreases its function in a dose-dependent manner as well as its ability to enhance phagocytosis of either gram-negative or gram-positive bacteria; 3) the activity of both SP-A1 and SP-A2 decreases in response to in vitro ozone exposure of proteins with SP-A2 being affected more than SP-A1. We conclude that both in vivo and in vitro oxidative modifications of SP-A by carbonylation reduce its ability to enhance phagocytosis of bacteria and that the activity of SP-A2 is affected more by in vitro ozone-induced oxidation. We speculate that functional differences between SP-A1 and SP-A2 exist in vivo and that the redox status of the lung microenvironment differentially affects function of SP-A1 and SP-A2.     

TGF-beta1 in SP-A preparations influence immune suppressive properties of SP-A on human CD4+ T lymphocytes

Surfactant protein A (SP-A) and transforming growth factor-beta1 (TGF-beta1) have been shown to modulate the functions of different immune cells and specifically to inhibit T lymphocyte proliferation. The aim of the present study was to elucidate whether the Smad signaling pathway, which is activated by TGF-beta1, also plays a role in SP-A-mediated inhibition of CD4+ T lymphocyte activation. Recombinant human SP-A1 expressed in Chinese hamster ovary cells [rSP-A1m (mammalian)], but not recombinant Baculovirus-derived rSP-A1hyp (hydroxyproline-deficient), suppressed T lymphocyte proliferation and IL-2 mRNA expression. To test whether SP-A induced Smad signaling, a Smad3/4-specific reporter gene was transfected in primary human CD4+ T lymphocytes. Only rSP-A1m, but not rSP-A1hyp, induced Smad-specific reporter genes, Smad2 phosphorylation, and Smad7 mRNA expression. The effect of rSP-A1m was mediated through the TGF-betaRII and could be antagonized by anti-TGF-beta1 neutralizing antibodies and sTGF-betaRII. Western blot and ELISA analysis revealed that rSP-A1m, but not rSP-A1hyp, contained TGF-beta1. TGF-beta1 was responsible for the differences in inhibition of CD4+ T lymphocyte proliferation and activation of the Smad signaling pathway between rSP-A1m and rSP-A1hyp. After acidification, native SP-A, obtained from patients with alveolar proteinosis, also induced Smad signaling in human CD4+ T lymphocytes leading to an increased inhibition of T lymphocyte proliferation, thus indicating the presence of inactive, latent TGF-beta1 in native SP-A samples. Association between SP-A and latent TGF-beta1 provides a possible novel mechanism to regulate TGF-beta1-mediated inflammation and fibrosis reactions in the lung but also leads to possible misinterpretation of immune-modulator functions of SP-A. Monitoring of SP-A preparations for possible TGF-beta1 is essential.     

SP-A1 and SP-A2 variants differentially enhance association of Pseudomonas aeruginosa with rat alveolar macrophages

Chronic airway inflammation caused by Pseudomonas aeruginosa is an important feature of cystic fibrosis (CF). Surfactant protein A (SP-A) enhances phagocytosis of P. aeruginosa. Two genes, SP-A1 and SP-A2, encode human SP-A. We hypothesized that genetically determined differences in the activity of SP-A1 and SP-A2 gene products exist. To test this, we studied association of a nonmucoid P. aeruginosa strain (ATCC 39018) with rat alveolar macrophages in the presence or absence of insect cell-expressed human SP-A variants. We used two trios, each consisting of SP-A1, SP-A2, and their coexpressed SP-A1/SP-A2 variants. We tested the 6A(2) and 6A(4) alleles (for SP-A1), the 1A(0) and 1A alleles (for SP-A2), and their respective coexpressed SP-A1/SP-A2 gene products. After incubation of alveolar macrophages with P. aeruginosa in the presence of the SP-A variants at 37 degrees C for 1 h, the cell association of bacteria was assessed by light microscopy analysis. We found 1) depending on SP-A concentration and variant, SP-A2 variants significantly increased the cell association more than the SP-A1 variants (the phagocytic index for SP-A1 was approximately 52-95% of the SP-A2 activity); 2) coexpressed variants at certain concentrations were more active than single gene products; and 3) the phagocytic index for SP-A variants was approximately 18-41% of the human SP-A from bronchoalveolar lavage. We conclude that human SP-A variants in vitro enhance association of P. aeruginosa with rat alveolar macrophages differentially and in a concentration-dependent manner, with SP-A2 variants having a higher activity compared with SP-A1 variants.     

Surfactant protein A binds to IgG and enhances phagocytosis of IgG-opsonized erythrocytes

Surfactant protein (SP)-A and SP-D, immunoglobulins, and complement all modulate inflammation within the lung by regulating pathogen clearance. For example, SP-A binds to and opsonizes a variety of bacteria and viruses, thereby enhancing their phagocytosis by innate immune cells such as alveolar macrophages. Immunoglobulins, which bind to antigen and facilitate Fc receptor-mediated phagocytosis, can also activate complement, a family of soluble proteins with multiple host defense functions. Previous studies showed that SP-A and complement protein C1q interact. Since complement protein C1q binds to IgG and IgM immune complexes, the hypothesis tested in this study was that SP-A, which is structurally homologous to C1q, also binds to IgG and affects its functions. SP-A binds to the Fc, rather than the Fab, region of IgG. Binding is calcium dependent but not inhibited by saccharides known to bind to SP-A's carbohydrate recognition domain. The binding of SP-A does not inhibit the formation of immune complexes or the binding of IgG to C1q. In contrast, SP-A enhances the uptake of IgG-coated erythrocytes, suggesting that SP-A might be influencing Fc receptor-mediated uptake. In summary, this study shows a novel interaction between SP-A and IgG and a functional consequence of the binding.     

SP-A binds alpha1-antitrypsin in vitro and reduces the association rate constant for neutrophil elastase

Background

α1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series of in vitro experiments aimed at investigating the nature and consequences of such an interaction.

Methods and results

At an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin.

Conclusion

We conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents.     

Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine

Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed. From these data we conclude that 1) SP-A specifically and strongly binds dipalmitoylphosphatidylcholine, 2) SP-A binds the nonpolar group of phospholipids, 3) the second positioned palmitate is involved in dipalmitoylphosphatidylcholine binding, and 4) the specificities of polar groups of dipalmitoylglycerophospholipids also appear to be important for SP-A binding, 5) the phospholipid binding activity of SP-A is dependent upon calcium ions and the integrity of the collagenous domain of SP-A, but not on the oligosaccharide moiety of SP-A. SP-A may play an important role in the regulation of recycling and intra- and extracellular movement of dipalmitoylphosphatidylcholine.     

Interaction of surfactant protein A with peroxiredoxin 6 regulates phospholipase A2 activity

Peroxiredoxin 6 (Prdx6) is a "moonlighting" protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA(2) activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA(2) activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA(2) activity) showed K(m)0.35 mm and V(max) 138 nmol/min/mg of protein. SP-A inhibited PLA(2) activity non-competitively with K(i) 10 mug/ml and was Ca(2+) -independent. Activity at pH 7.4 was approximately 50% less, and inhibition by SP-A was partially dependent on Ca(2+). Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni(2) -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca(2+) dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA(2) activity of Prdx6 by SP-A.     

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).     

Differences in biochemical properties and in biological function between human SP-A1 and SP-A2 variants, and the impact of ozone-induced oxidation

The human surfactant protein A (SP-A) locus consists of two functional genes, SP-A1 and SP-A2, with several alleles characterized for each gene. Functional variations between SP-A1 and SP-A2 variants either before or after ozone exposure have been observed. To understand the basis of these differences, we studied SP-A1 and SP-A2 variants by comparing coding sequences, oligomerization patterns under various conditions, composition of oligomers with regard to amino terminal sequence isoforms, biological activity (regulation of phosphatidylcholine (PC) secretion by alveolar type II cells), and the impact of ozone-induced oxidation. We found that (i) the SP-A1 (6A(4)) allele is the most divergent from all SP-A2 alleles, particularly from the SP-A2 (1A(1)). (ii) Differences exist in oligomerization among SP-A1, SP-A2, and coexpressed SP-A1/SP-A2, with higher order multimers (i.e., consisting of more subunits) observed for SP-A1 than for SP-A2 variants. Differences among SP-A1 or SP-A2 gene products are minimal. (iii) Amino acid variants in the amino terminal sequences are observed after signal peptide removal, including variants with an extra cysteine. (iv) Oxidation is observed after ozone exposure, involving several SP-A residues that include cysteine, methionine, and tryptophan. (v) The SP-A2 variant (1A(0)) and the coexpressed protein 1A(0)/6A(2) inhibit ATP-stimulated PC secretion from alveolar type II cells to a greater extent than SP-A1 (6A(2)), a biologic activity that was susceptible to ozone treatment.     

Pulmonary collectins enhance phagocytosis of Mycobacterium avium through increased activity of mannose receptor

Collectins, including surfactant proteins A (SP-A) and D (SP-D) and mannose binding lectin (MBL), are the important constituents of the innate immune system. Mycobacterium avium, a facultative intracellular pathogen, has developed numerous mechanisms for entering mononuclear phagocytes. In this study, we investigated the interactions of collectins with M. avium and the effects of these lectins on phagocytosis of M. avium by macrophages. SP-A, SP-D, and MBL exhibited a concentration-dependent binding to M. avium. The binding of SP-A to M. avium was Ca(2+)-dependent but that of SP-D and MBL was Ca(2+)-independent. SP-A and SP-D but not MBL enhanced the phagocytosis of FITC-labeled M. avium by rat alveolar macrophages and human monocyte-derived macrophages. Excess mannan, zymosan, and lipoarabinomannan derived from the M. avium-intracellular complex, significantly decreased the collectin-stimulated phagocytosis of M. avium. Enhanced phagocytosis was not affected by the presence of cycloheximide or chelation of Ca(2+). The mutated collectin, SP-A(E195Q, R197D) exhibited decreased binding to M. avium but stimulated phagocytosis to a level comparable to wild-type SP-A. Enhanced phagocytosis by cells persisted even after preincubation and removal of SP-A or SP-D. Rat alveolar macrophages that had been incubated with SP-A or SP-D also exhibited enhanced uptake of (125)I-mannosylated BSA. Analysis by confocal microscopy and flow cytometry revealed that the lung collectins up-regulated the cell surface expression of mannose receptor on monocyte-derived macrophages. These results provide compelling evidence that SP-A and SP-D enhance mannose receptor-mediated phagocytosis of M. avium by macrophages.     

Characterization of a human surfactant protein A1 (SP-A1) gene-specific antibody; SP-A1 content variation among individuals of varying age and pulmonary health

The human surfactant protein A (SP-A) locus consists of two functional genes (SP-A1, SP-A2) with gene-specific products exhibiting qualitative and quantitative differences. The aim here was twofold: 1) generate SP-A1 gene-specific antibody, and 2) use this to assess gene-specific SP-A content in the bronchoalveolar lavage fluid (BALF). An SP-A1-specific polyclonal antibody (hSP-A1_Ab(68-88)_Col) was raised in chicken, and its specificity was determined by immunoblot and ELISA using mammalian Chinese hamster ovary (CHO) cell-expressed SP-A1 and SP-A2 variants and by immunofluorescence with stably transfected CHO cell lines expressing SP-A1 or SP-A2 variants. SP-A1 content was evaluated according to age and lung status. A gradual decrease (P < 0.05) in SP-A1/SP-A ratio was observed in healthy subjects (HS) with increased age, although no significant change was observed in total SP-A content among age groups. Total SP-A and SP-A1 content differed significantly between alveolar proteinosis (AP) patients and HS, with no significant difference observed in SP-A1/SP-A ratio between AP and HS. The cystic fibrosis (CF) ratio was significantly higher compared with AP, HS, and noncystic fibrosis (NCF), even though SP-A1 and total SP-A were decreased in CF compared with most of the other groups. The ratio was higher in culture-positive vs. culture-negative samples from CF and NCF (P = 0.031). A trend of an increased ratio was observed in culture-positive CF (0.590 +/- 0.10) compared with culture-positive NCF (0.368 +/- 0.085). In summary, we developed and characterized an SP-A1 gene-specific antibody and used it to identify gene-specific SP-A content in BALFs as a function of age and lung health.     

Pollen grains bind to lung alveolar type II cells (A549) via lung surfactant protein A (SP-A)

Lung surfactant protein A (SP-A) is the most abundant surfactant-associated protein present in the lung. A receptor for SP-A has been shown to be present on A549 alveolar type II cells and on other cell types, including alveolar macrophage. The SP-A receptor on A549 cells has been identified as the collectin receptor, or C1q receptor, which binds several structurally-related ligands. SP-A contains C-type lectin domains, but the role of carbohydrate binding by SP-A in physiological and pathological phenomena is not yet established. In this paper we report the binding of SP-A to pollen from Populus nigra italica (Lombardy Poplar), Poa pratensis (Kentucky blue grass),Secale cerale (cultivated rye) and Ambrosia elatior (short ragweed). Saturable and concentration dependent binding of SP-A to pollen grains was observed. Interaction of SP-A with pollen grains takes place through waterextractable components, in which the major species present, in Lombardy poplar pollen,are 57 kD and 7 kD (glyco)proteins. The binding of SP-A to pollen grains and their aqueous extracts was calcium ion dependent and was inhibited by mannose, and is therefore mediated by the lectin domain. Binding of SP-A to pollen grains was found to mediate adhesion of pollen grains to A549 cells. The results suggest that pollen grains or other carbohydrate-bearing particles (e. g. microorganisms) could potentially interact with different cell types via the collectin receptor (C1q Receptor) in the presence of SP-A.